THE MECHANISM OF THE NEPHRIDIAL APPARATUS OF MUL TIMICRONUCL EATUM

1. Expulsion of Water from the Vesicle

ALAN E. ORGAN, EUGENE C. BOVEE, THEODORE L. JAHN, Downloaded from http://rupress.org/jcb/article-pdf/37/1/139/1384276/139.pdf by guest on 30 September 2021 DUNCAN WIGG, and JAMES R. FONSECA

From the Department of Zoology, University of California, Los Angeles, California 90024

ABSTRACT Recent analysis of the mechanism of the nephridial apparatus of Paramecium multimicronu- cleatum by high-speed cinematography (300 fps at X 250) confirms the observations by electron microscopy (Schneider, 1960) that once the pore is opened, the vesicle is invaginated by adjacent cytoplasm and is emptied by collapsing under pressure from that cytoplasm, aided perhaps by pressure of the fibrils which anchor the ampullae to the excretory canal. There is no indication of active contraction of the vesicle or its membrane. There is no permanent pore to the vesicle. The vesicle is closed by a sealing of the ruptured membrane where it is in contact with the pellicular excretory canal. At onset of expulsion of vesicular fluid the membrane across the basal opening of the excretory canal is ripped along one semicircular portion of the excretory pore and is driven up against the opposite wall as a flap while the water rushes out. A constriction of the vesicular and cell membranes at the base of the excretory canal reseals the opening.

The discovery of the water-expulsion vesicle contributory function. While no direct measure- ("contractile vacuole") of Paramecium is usually ments are known for the pressure required to attributed to Joblot in 1718, and the observation empty the vesicle, Kitching calculates a necessary of the repeated appearance of the ampullary hydrostatic pressure of 0.33 cm of water greater canals of Paramecium attributed to Spallanzani in within the vesicle than outside it to empty it 1776. These vesicles are and have been repeatedly (Kitching, 1952). Such a pressure he assumes may the basis for study in protozoa dealing with their possibly develop as a result of tension within or development and structure and their functions in pressure upon the vesicular membrane. He further filling and expulsion (see references: von Gelei, assumes that, in at least, the pressure is the 1935; Kitching 1952, 1954, 1956, 1967; Lloyd, result of tension in the vesicular wall which causes 1928, Weatherby, 1941). Unfortunately, little the wall to contract during evacuation. He ad- critical attention has been given to the mechanism mits, however, that there is no knowledge of by which the vesicle is emptied. whether an oriented protein layer around the While turgor is still considered by some, e.g. vesicle is present or is involved in the process Czarska, 1964, to be involved in the expulsion, (Kitching, 1956). This assumed contractility of Kitching (1956, 1967) dismisses body turgor as the water-excretory vesicle of protozoa has been being essential to expulsion of the vesicle (described generally accepted; ever since the studies of as a "systole"), and he assigns it only a possible Claparede (1854) and Lachmann (1859) the

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FIGURE 1

FIGURE 1 Photomicrographs from a motion picture (300 fps) showing the rupture and evacuation of the water-expulsion vesicle of Parameciummultimicronucleatum (side view). In frame 1, the pore-sealing mem- brane across the basal end of the excretory canal (internal pore) is still intact. Frames 2-6 show the ripping of that membrane and the subsequent opening of the internal pore. Frames 9-35 show the invagination of the bottom of the vesicle and the emptying of it. In frame 36, the cytoplasm beneath the internal pore of the excretory canal has constricted and formed a new pore-sealing membrane resealing the old vesicle. X 1600. Downloaded from http://rupress.org/jcb/article-pdf/37/1/139/1384276/139.pdf by guest on 30 September 2021

FIGURE

FIGURE 2 Photomicrographs from a motion picture (300 fps) showing the same sequence of the opening of the pore-sealing membrane as Fig. 1, but from a surface view. The pore, at first dark in the photograph, appears white as it opens. X 3200.

FIGURE

FIGURE 3 Sketches based on selected tracings of photomicrographs in Fig. 1 showing the invagination of the vesicle. The dotted line in the lower portion of frames 12-36 denotes the position of the vesicular mem- brane before evacuation started. structure has been and still is usually termed the For example, Taylor (1923) wrote of the water- contractile vacuole, not only in ciliates but in other excretory vesicle of patella as collapsing, taxonomic groups. and older drawings by Faur6-Fremiet (1905) of However, there is some contradictory evidence. Campanella umbellaria show a collapse of the vesicu-

A. E. ORGAN, E. C. BVEE, T. L. JAIN, D. WIGG, AND J. R. FONSECA Nephridial Apparatus 141 Downloaded from http://rupress.org/jcb/article-pdf/37/1/139/1384276/139.pdf by guest on 30 September 2021 FIGRE 4 FIGURE 4 The frames from Figs. 1 and 2, that are equivalent, are shown together to clarify the sequence of the opening of the pore-sealing membrane from both views. In the lower row the membrane appears dark, and the open pore appears light. In frame 7 the flap is assumed to be the dark area at the lower left side of the pore. This dark area does not appear in frames 6 or 8 or in other frames of the same sequence. X 1600.

FIGURE 5 FIGURE 5 Sketches based on Fig. 4. lar membrane against itself in ciliates. Furthermore, MATERIALS AND METHODS older observations by Penard (1902) and Metcalf Paramecium multimicronucleatum (Gittleson strain), (1910), as well as recent cinephotomicrographs of grown on lettuce medium and buffered to a pH of the water expulsion vesicle of Amoeba proteus by 7.2 with CaCO 3 , was studied. The lettuce medium Wigg et al. (1967), show that the water expulsion was made with Chalkley's solution and was inoculated vesicle of Amoeba proteus does not contract. These with Bacillus subtillus. Vigorous and numerous studies and others to be mentioned later have led paramecia were thus available at any time during us to reinvestigate the evacuation of the water- these studies. The observed paramecia were im- excretory vesicle in Paramecium multimicronucleatum mobilized on No. 1 thickness microscope slides coated by cinephotomicrography. with an even, thin layer of 1% nonnutrient agar.

142 THE JOURNAL OF CELL BIOLOGY VOLUME 37, 1968 FIGURE 6 FIGURE 6 Top (surface) view of the evacuation of the water expulsion vesicle of Paramecium multi- micronucleatum. Shows that the vesicle retains its circular shape in the horizontal plane as the bottom of vesicle becomes invaginated, and the vesicle decreases in volume. The frames shown are numbers 8, 28, 38, 48, and 58 respectively of the same sequence as the lower row of Fig. 4. X 2400. Downloaded from http://rupress.org/jcb/article-pdf/37/1/139/1384276/139.pdf by guest on 30 September 2021

The paramecia adhered by the tips of their cilia and 12-36), and it appears to collapse under the were thus just immobilized, but otherwise normal. pressure created by the adjacent cytoplasm and The evacuatory movements of the water-excretory perhaps some pressure created by the fibrils which vesicle were repeatedly photographed in the same anchor the ampullae to the excretory canal. and in different, individual paramecia for determina- At the onset of expulsion of vesicular fluid, the tion of the nature and mechanism of the movements, and their sequences, involved in emptying the vesicle. pore-sealing membrane across the basal opening Fastair and Hycam 16-mm motion picture cameras of the excretory canal is ripped along one semi- with variable framing rates (up to 500 fps) were used circular border, and is driven up against the with a Carl Zeiss variable phase contrast microscope opposite wall as a flap while the water rushes out at X 250 magnification. The light source was a (Fig. 1, frames 1-5; Fig. 2, frames 1-8; and Fig. 3, carbon arc lamp. Motion pictures were taken at in which the upper row represents stages seen in 300 fps. The film was professionally developed and Fig. 1 and the lower row those from Fig. 2). printed. Fig. 4 shows frames from Figs. 1 and 2 which are Analysis of the evacuatory movements was made equivalent in order to clarify the sequence of the by repeated viewing of the films at varied framing opening of the pore-sealing membrane from both rates (2-24 fps) with a flickerless L-W Photo-optical analyzer and by frame by frame analysis of the single the side and surface views Fig. 5 is a series of pictures printed on Kodak Polycontrast paper (East- diagrams based on the photographs of Fig. 4. man Kodak Co., Rochester, N.Y.). The membrane opens approximately 15 msec before the expulsion of the vesicular fluid by OBSERVATIONS means of cytoplasmal pressure which can be As has been suggested by the electron micrography detected in the photographs. After the pore opens, done by Schneider (1960) on paramecia and by the top (outermost) portion of the vesicle becomes Mosevitch (1965) on Ichthyophthirius multifiliis, the rigid as the pressure of the cytoplasm causes the water-excretory vesicle of Paramecium multimicro- vesicle to invaginate upon itself. Initially, as the nucleatum is collapsed during its evacuation and vesicle commences its expulsion, there is a small does not of itself contract. pressure exerted by the fibrils which are attached The evacuation of the vesicle is shown photo- to the ampullae and wind over the top hemisphere graphically in Figs. 1 and 2, and semidiagrammati- of the vesicle. As the expulsion continues, the cally in Fig. 3. The photographs are a sequence of pressure exerted by those fibrils increases as the frames from a motion picture which clearly shows fibrils are stretched while the ampullae fill with the evacuatory process which takes only milli- more water. seconds for completion. Our best estimates, based Contrary to the description by King (1935) in on several sequences, indicates an average time of his observations of the expulsion of fluid from the 108.89 msec, or about o0 sec. vesicle in P. multimicronucleatum, the bottom of the The vesicle becomes invaginated by adjacent old contractile vacuole does not form the new cytoplasm (Fig. 1, frames 9-35; Fig. 3, frames membrane over the pore as it is pushed up toward

A. E. ORGAN, E. C. BOVEE, T. .. JAHN, D. WIGG, AND J. R. FONSECA Nephridial Apparatus 143 the pore. Actually, the bottom of the membrane (Schneider, 1960), pyriformis (Elliott never reaches the basal part of the excretory canal. and Bak, 1964), and Ichthyophthirius multifiliis King's statement was based on visual observation (Mosevitch, 1965) explicitly show an invagination of a phenomenon that we now know is too fast for of the vesicle during expulsion. This invagination the eye to follow. In Fig. 1, frames 6-42 show the is probably due to movements of the endoplasmic lower (innermost) margin of the vesicle moving gel pushing on the vesicular membrane. All of the upward; but it almost stops approximately by above authors except Elliott and Bak (Tetrahy- frame 36 and advances very little in frames 36- mena) refer to the vesicle as collapsing rather than 42. It never reaches the position of the pore-sealing contracting. Metcalf (1910) referring to the water- membrane shown in Fig. 1, frame 1. This is shown expulsion vesicle of Amoeba proteus, and Taylor diagrammatically in Fig. 3. (1923) describing the water-expulsion vesicle of As the pressure of the vesicular fluid content Euplotcs, also indicate a collapse of the vesicle. All flowing through the excretory canal is decreased, of the above investigators, with the exception of the flap that covered the excretory canal may fall Elliott and Bak, interpret the evacuation of the back into place. Then a membrane at the basal vesicle as due to force developed in the movements Downloaded from http://rupress.org/jcb/article-pdf/37/1/139/1384276/139.pdf by guest on 30 September 2021 end of the pore, which looks like a constriction of of the endoplasmic gel, and not to contraction of the pore canal, reseals the opening. This apparent the vesicle itself. Elliott and Bak (1964) invoke constriction membrane probably consists of the the action of unidentified fibrils and suggest that marginal area of the original pore-sealing mem- these fibrils have only a "facilitating" function brane, including the flap, and is not a constriction while implying that expulsion requires another in the sense of a contraction of the wall of the pore principal mechanism. canal. After this has occurred, the ampullae refill If discrete fibrils are involved in a contraction of the vesicle. This constriction has completely the vesicle, as Elliott and Bak (1964) suggest, closed the pore in frames 37-42 and has partially then, in electron micrographs they should be ob- blocked the pore in frames 32-36 and to a lesser servable surrounding the vesicle. According to the extent in earlier frames. electron micrographs of Schneider (1960) many As the vesicle is emptied it remains circular, as (20-30) long fibrils form a spiral band from each seen in surface view, at least until its volume is ampulla and its injection tubule. These fibrils reduced by more than 50 %. This is shown in Fig. wind clockwise over that hemisphere of the vacuo- 6, a sequence of surface views, approximately lar cavity which is adjacent to the pellicular pore; equivalent to Fig. 3, frames 1-16. In this sequence, then they wind internally also around the short volume reduction is brought about mostly by cylindrical tube of the pellicular pore and fasten invagination of the innermost wall of the vesicle to the pore and inner surface of the pellicle where (shown in Fig. 3) and only secondarily by a reduc- the latter invaginates to form the pore. There are tion in diameter of the vesicle (Fig. 6). After the also some fibrils adjacent to the membrane of the above-mentioned frames, the vesicle went out of ampullary segment of the tubule, but there are no focus; so it was impossible to determine accurately discernible fibrils in or adjacent to the internally whether the vesicle stayed circular throughout the hemispheric segment, when filled, of the vesicular evacuation. However, the impression still remains space. that the vesicle does remain circular throughout as It is evident that the ampullary fibrils associ- seen in frames 25-36 of Fig. 1. There is no indica- ated with the expulsion vesicle of Paramecium tion of active contraction of the vesicle or its multimicronucleatum cannot be wholly responsible for membrane. a "contraction" of the vesicle. It is now obvious, from our observations and from evidence in the DISCUSSION literature, that the water-excretory vesicle of Electron micrographs and reconstruction diagrams Paramecium multimicronucleatum is not a truly con- of the movements of the water-expulsion vesicle of tractile organelle. Since the water-excretory vesicle Amoeba proteus (Penard, 1902; Metcalf, 1910; Wigg of P. multimicronucleatum does not contract, it can- et al., 1967), Campanella umbellaria and Ophrydium not properly be called a contractile vacuole. As versatile (Faur6-Fremiet, 1905; FaurE-Fremiet and already has been proposed (Wigg et al., 1967), we Rouiller, 1959), Tokophrya infusionum (Rudzinska, also propose that a better term for it might be the 1958), Paramecium aurelia and Paramecium caudatum water-expulsion vesicle, since any method of expul-

144 TIIE JOURNAL OF CELL BIOLOGY VOLUME 87, 1968 sion is in accord with that term. We also believe the flap. Therefore, evidence of existence of the that the term "systole," which implies contraction, flap does not consist entirely of the present photo- might better be replaced by the term evacuation, graphs, which, alone, may not be entirely con- and "diastole" by enlargement; thus we would avoid vincing. However, the flap can be seen better in the implication of a repetitive cycle of contraction the original motion picture when projected than in and relaxation which does not exist. single frames, and also it can be deduced from The pore-sealing membrane, which breaks on Schneider's electron micrographs. one side and thereby becomes a flap (Fig. 3, frames 1-6), is shown clearly in the electron This research was supported by grant 6462 from the micrograph of Schneider (1960, Fig. 8 a). Schnei- National Institutes of Health, grants GB 1589 and der's figure shows the membrane closed; the area GB 5573 from the National Science Foundation, and in the center of the pore canal is very thin, while Office of Naval Research contract NONR 4756. Alan E. Organ is a National Science Foundation that near the pore wall is much thicker, especially researcher under the auspices of the Committee for on one side, and irregular in outline. Our assump- Advance Science Training. Downloaded from http://rupress.org/jcb/article-pdf/37/1/139/1384276/139.pdf by guest on 30 September 2021 tion is that the thin area breaks under pressure Received for publication 5 October 1967, and in revisedform and that the thicker portion is pushed outward as 5 December 1967.

REFERENCES

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A. E. ORGAN, E. C. BOVEE, T. L. JAHN, D. WIGG, AND J. R. FONSECA Nephridial Apparatus 145