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Chapter 31 Reactivation of Extracted Paramecium Models
CHAPTER 31
Reactivation of Extracted Paramecium Models Yutaka Naitoh Pacific Biomedical Research Center and Department of Microbiology University of Hawaii at Manoa Honolulu. Hawaii 96822
I. Introduction 11. Methods
A. Triton X-100 Extraction of Paramecium B. Modified Triton X-100 Extraction of Paramecium C. Glycerol Extraction of Paramecium D. Triton-Glycerol Extraction of Paramecium E. Modified Triton-Glycerol Extraction of Paramecium F. Isolation of Ciliated Cortical Sheets from Triton-Extracted Paramecium References
I. Introduction The swimming behavior of Paramecium is dependent mostly on its ciliary motile activity. Ciliary motile activity is regulated by membrane electrogenesis (Naitoh and Eckert, 1969; Eckert, 1972, Naitoh, 1982). A depolarizing receptor potential evoked by stimulation of the front end of Paramecium electrotonically spreads over the entire cell membrane to activate (open) depolarization-sensitiveCa2+channels in the ciliary membrane. Activation of the channels causes regenerative entry of Ca2+ ions into the cilia, producing a sudden increase in the intraciliary Ca2+concentration and an action potential. The Ca2+increase activates a mechanism for reversing the direction of the effective power stroke of cilia to cause backward swimmingof Paramecium. Paramecium thereby avoids a noxious stimulus at its front. METHODS IN CELL BIOLOGY,VOL. 47 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved
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A hyperpolarizing receptor potential evoked by stimulation of the posterior end of Paramecium also spreads electrotonically over the entire cell membrane. Hyperpolarization of the membrane brings about an increase in the intracellular CAMP concentration, which activates the ciliary beating mechanism to cause an increase in the frequency of ciliary beating in the normal direction (Nakaoka et al., 1984; Majima et al., 1986; Bonini et al., 1986). This brings about rapid forward swimming of the specimen. Paramecium thereby escapes from a noxious stimulus at its rear. To understand the mechanism by which ciliary motile activity is regulated by these chemical messengers, it is important to examine the direct effects of the chemicals on the motile activity of the ciliary apparatus. An external application of these chemicals does not, however, necessarily increase the intracellular concentrations of the chemicals, as the cell membrane is poorly permeable to these chemicals. Moreover, an external application of Ca2+ions will even produce a decrease in the intracellular Ca2+concentration in Paramecium, because it deactivates Ca2+channels so that Ca2+influx decreases (Naitoh, 1981). Therefore, permeabilization of the cell membrane is one method essential for examining the direct effects of the chemicals. In a permeabilized cell, functions of the membrane as a signal transducer and a diffusion barrier are disrupted, while the motor function of the ciliary apparatus is kept normal. The idea of permeabilization of the cell originated from a classic work on the glycerinated muscle fiber by Szent-Gyorgyi (1947). Hoffman-Berling (1955) applied Szent-Gyorgyi’s technique to protozoan cells to make “ciliary models.” Gibbons and Gibbons (1972) first introduced a nonionic detergent, Triton X100, to obtain permeabilized seaurchin spermatozoa so good that almost 100% of them showed ATP-Mg2+-reactivated flagellar motion. It should be noted that a detergent destroys not only the membrane but also the ciliary apparatus. The concentration of the detergent, the time of exposure of the cells to the detergent, the ambient temperature, and the ionic conditions all influence the quality of the extracted models. Tolerance to a given detergent differs from specimen to specimen depending on their culture condition, cell size, species, and strains. Although Triton X100 is the most popular detergent for permeabilization of Paramecium, it is worthwhile examining other detergents as well, such as Nonidet P-40 (Goodenough, 1983) and saponin (Seravin, 1961). In addition to ATP, Mg2+, and KCl, other chemicals in the reactivation solution are important to keep the reactivated ciliary activity stable. Acetate ions, instead of C1- (Gibbons et al., 1982), and polyethylene glycol (Goodenough, 1983) have been used to stabilize the ciliary activity. It is important to determine your own conditions for permeabilization adequate for your experimental purposes. Therefore, the recipes given in the following sections are subject to your own modification.
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11. Methods A. Triton X-100 Extraction of Paramecium
This standard procedure of Naitoh and Kaneko (1972) for Triton X-100 extraction of Paramecium caudatum has been employed, with minor modifications, by many authors for other species of Paramecium (P.tetraurelia: Kung and Naitoh, 1973; Bonini et al., 1986; Lieberman et al., 1988; P . multimicronucleaturn: Okamoto and Nakaoka, 1994) and other genera of ciliates, including Tetrahymena (Takahashi et al., 1980; Goodenough, 1983) and Euplotes (Epstein and Eckert, 1973).
1. Culture of Specimens Specimens obtained from any kind of culture (bacterized hay, rice straw, wheat straw, lettuce, or milk infusion) can be used. Crude cultures, such as bacterized hay infusion made over a layer of field soil in a ceramic bowl, are preferable to the more sophisticated axenic cultures to obtain good extracted specimens. Such specimens show coordinated and long-lasting ciliary movement when reactivated. Axenic culture, however, is essential for more critical biochemical examinations of the extracted specimens.
2. Stock Solutions 0.1 M KCI 0.1 M CaCI, 0.1 M MgCl, 0.1 M EDTA (neutralized by KOH) 0.1 M EGTA (neutralized by KOH) 0.1 M Tris-maleate pH buffer (PH 7.0)
0.1 M Tris-HC1 pH buffer (pH 7.4) 0.1 M ATP (neutralized by KOH, to be stored in a freezer) 0.1 M KOH 0.1 M HC1 1% (by volume) Triton X-100
3. Experimental Solutions Washing solution I (for living specimens from culture): 2 mM CaCI, + 1 mM Tris-HC1 (pH 7.2) (in final concentration). Mix 10 ml of 0.1 M CaCI,, with 5 ml of 0.1 M Tris-HC1 and add H,O to make 500 ml of solution. Extraction solution: 20 mM KCI + 10 mM ethylenediaminetetraacetic acid (EDTA) + 10 mM Tris-maleate (pH 7.0) + 0.01% (by volume) Triton X-100. Mix 100 ml of 0.1 M KC1, 50 ml of 0.1 M EDTA, 50 ml of 0.1 M Tris-maleate (pH 7.0), and 5 ml of 1% Triton X-100, and add H,O to make 500 ml of solution.
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Washing solution I1 (for extracted specimens): 50 mM KCl + 10 mM Tris-maleate (pH 7.0). Mix 250 ml of 0.1 M KCl with 50 ml of 0.1 MTris-maleate and add H 2 0 to make 500 ml of solution. Test solutions: Basic components of the test solutions are the same as those of the washing solution 11. Chemicals to be tested are added to the solution. Two representative test solutions are:
1. Standard reactivation solution: 50 mM KC1 + 4 mM MgCl, + 4 mM ATP + 3 mM ethylene glycol bis (B-aminoethyl ether)-N,N-tetraacetic acid (EGTA) + 10 mM Tris-maleate (pH 7.0). Mix 5 ml of 0.1 M KCl, 0.4 ml of 0.1 M MgCl,, 0.4 ml of 0.1 M ATP, 0.3 ml of 0.1 M EGTA, and 1 ml of 0.1 M Tris-maleate and add H,O to make 10 ml of solution. The extracted specimens swim forward in this solution. 2. Ca series reactivation solutions: Add an amount of 0.1 M CaCl, to be determined by the experiment to the standard reactivation solution. The concentration ratio of Ca2+to EGTA determines the free Ca2+concentration in the solution (Ca2+buffer; Portzehl et ul., 1964;Noguchi et ul., 1986).The extracted specimens swim backward when the free Ca2+concentration in the solution is above M.
4. Experimental Procedures a. Collecting and Washing the Specimens
Introduce about 20 ml of culture medium containing specimens of Purumecium into a long-necked flask (such as a 50-ml volumetric flask). Fill the flask with washing solution I. Keep the flask still on a table for ca. 4 minutes. Specimens will gather at the neck portion of the flask due to their negative geotaxis. Pipet the specimens into another long-necked flask, then fill the flask with the washing solution. The specimens again gather in the neck portion. Repeat this procedure three times to dilute the culture medium. Pipet the concentrated specimens in the neck portion into a centrifuge tube. Keep the tube in an ice bath for 3 minutes. Centrifuge the tube gently to make a loose pellet of the specimens. Remove the supernatant by suction. 6. Extracting the Specimens Introduce ca. 5 ml of cold (0-1°C) extraction solution into the tube to suspend the washed specimens in the solution. Keep the tube vertical in an ice bath for ca. 30 minutes. Specimens gradually lose their mobility in the extraction solution and sink toward the bottom of the tube. Remove the extraction solution by suction, leaving the extracted specimens at the bottom. c. Washing the Extracted Specimens
Introduce ca. 5 ml of cold (0-l°C) washing solution I1 into the tube to suspend the extracted specimens in the solution. Centrifuge the tube very gently to
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make a loose pellet of the specimens. Remove the supernatant by suction. Repeat this procedure three times to wash the Triton X-100and EDTA away from specimens. Keep the extracted specimens cooled in an ice bath, in a minute amount of the washing solution. d. Reactivating the Extracted Specimens Pipet about 1 pl of washing solution containing hundreds of extracted specimens into ca. 1 ml of a test solution on a depression slide at room temperature (ca. 24°C). Stir the mixture gently. A few seconds after this mixing, the extracted specimens will begin to swim due to the reactivation of the ciliary movement, providing the test solution contains an adequate amount of ATP and Mg2+. e. Observing and Recording Reactivated Specimens Pipet about 100 pl of test solution containing scores of reactivated specimens onto a glass slide to make a droplet. Put several small pieces of crushed coverslip into the droplet so that a thin space is made when a coverslip is put on the droplet. The extracted specimens will swim freely in the thin space between the glass slide and the coverslip. Put white vaseline around the edge of the coverslip to prevent evaporation of the test solution. The specimens are now ready for observation and recording of their motile activity. Swimming Paths of the Reactivated Specimens. Under dark-field illumination, swimming specimens are easily observed through a conventional binocular dissecting microscope with a final magnification of x 10- x 20. Movement of the specimens can be recorded with a conventional video recorder. The swimming path of a specimen is then determined by frame-by-frame analysis of displayed images of the specimen. Various kinds of computerized analyzers are available for this type of analysis (e.g., see Chapter 40 in this volume). The swimming path of a specimen can be photographed as a curved spiral line on a still film when the exposure time is extended to 1-5 seconds. Swimming direction and swimming velocity can be determined from the swimming path. Ciliary Motile Activity. A conventional phase-contrast, interferencecontrast, or dark-field microscope with a final magnification of X 200- X 400 is essential for observing and recording the reactivation of the cilia. High-speed video recording of the cilia is essential to determine the beat frequency, the direction of the effective power stroke, and the beating form of an individual cilium (see Chapter 34 in this volume). The computerized image contrastenhancement technique is useful for obtaining a good image of a cilium (see Chapter 38 in this volume). The beat frequency of reactivated cilia of an extracted specimen can be determined from the frequency of change in light intensity of a selected small area of a magnified image of the specimen due to the passage of metachronal waves. This is accomplished by placing a photodiode or a photomultiplier in the path of the image (Naitoh and Kaneko, 1973). The relative force exerted by the reactivated cilia on a given specimen can
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be estimated from the swimming path of the specimen (Naitoh and Sugino, 1984). The direction of the force corresponds to the mean direction of the effective power strokes, and the amount of force is proportional to the mean beat frequency of all the cilia. Ciliary metachronal waves can be observed as darker (or brighter) lines moving on the ciliary surface or as waves moving along the margin of a reactivated specimen. The metachronal wave lines are easily photographed on film. The direction of the effective power stroke can be determined from the metachronal wave line, as the angle between the metachronal wave line and the direction of the effective power stroke is always 90"(Machemer, 1972). The direction in which nonbeating cilia point, which usually corresponds to the direction of the effective power stroke, can be determined from a photograph of an extracted specimen.
B. Modified Triton X-100 Extraction of Paramecium Nakaoka and Toyotama (1979) added Mg2+ ions to the Triton X-100containing extraction solution. Specimens extracted with this solution showed higher sensitivity to Ca2+in modifying beat frequency in Mg2+-ATP-reactivated cilia than those extracted with a solution without Mg2+.The maximum beat frequency under optimum reactivation conditions was higher in the Mg2+containing extraction solution and close to the maximum beat frequency of living specimens. Formation of metachronal waves on the reactivated ciliary surface was also enhanced (Nakaoka et al., 1984; Nakaoka and Ooi, 1985). The presence of Mg2+ in the extraction solution maintains the ciliary motile mechanisms of Paramecium in a closer-to-normal condition than when Mg2+ is absent during the Triton X-100 treatment.
1. Experimental Solutions Extraction solution: 20 mM KCl + 5 mM MgCl, + 5 mM EGTA + 0.008% (by volume) Triton X-100 + 10 m M Tris-maleate buffer (pH 7.0) (in final concentration). Washing solution I: 20 mM KCl + 5 mM MgC1, + 3 mM EGTA + 10 mM Tris-maleate buffer (pH 7.0). Washing solution 11: 20 mM KCl + 5 mM MgCl, + mM Tris-maleate buffer (pH 7.0). Reactivation solutions: 10 mM KC1 + 8 mM MgCl, + 4 mM ATP + 1 mM EGTA + an amount of CaC1, needed to provide the appropriate free Ca2+ concentration required for the experiment + 10 mM Tris-maleate buffer (pH 7.0).
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2. Experimental Procedures a. Collecting and Washing Cultured Specimens See Section II,A,4,a.
b. Extracting the Specimens Suspend the washed specimens in the extraction solution in a centrifuge tube for 10-12 minutes at room temperature (25°C). Remove the extraction solution by suction, leaving the extracted specimens at the bottom of the tube. c. Washing the Extracted Specimens Introduce cold (OOC) washing solution I into the tube to suspend the extracted specimens in the solution. Keep the tube vertical in an ice bath for 15 minutes. The extracted specimens will sink toward the bottom of the tube. Remove the washing solution by suction, leaving the specimens at the bottom of the tube. Introduce cold (OOC) washing solution I1 into the tube to suspend the specimens. Keep the tube in an ice bath. The specimens sink toward the bottom in 15 minutes. Washed specimens thus obtained are ready for experimentation. d. Reactivating the Extracted Specimens Pipet a concentrated suspension of the extracted specimens into ca. 1 ml of a reactivation solution at room temperature (20-22°C). Stir the mixture gently for a few seconds. Observations and measurements are made 3-20 minutes after mixing at room temperature.
C. Glycerol Extraction of Paramecium Glycerol-extracted Paramecium models do not show ciliary beating in the presence of ATP and Mg2+; however, glycerol extraction supports a change in the direction in which nonbeating cilia point on administration of ATP and Ca2+. That is, cilia swing once to change their pointing direction (ciliary reorientation response). The reorientation response in nonbeating cilia corresponds to a change or reversal of the direction of the effective power stroke (ciliary reversal) in beating cilia (Naitoh, 1966). Glycerol, therefore, specifically destroys the beating mechanism in the ciliary motile apparatus.
1. Experimental Solutions
Washing solution I (for specimens from culture): 2 mM CaCl, + 1 mM Tris-HC1 (pH. 7.2) (in final concentration). 10 mM Tris-HC1 Extraction solution: 50 mM KC1 10 mM EDTA (pH 7.4) + 50% (by volume) glycerol.
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Washing solution I1 (for extracted specimens): 50 mM KCl + 10 mM Tris-HC1 (PH 7.4). Test solutions: Basic components of a test solution are the same as those of the washing solution I1 except for pH (pH is 9.0 instead of 7.4 in the washing solution). Chemicals to be tested are added to the basic solution.
2. Experimental Procedures a. Collecting and Washing the Cultured Specimens See Section II,A,4,a.
b. Extracting the Specimens Introduce ca. 5 ml of cold (0-1°C) extraction solution into the centrifuge tube containing a pellet of washed specimens, and stir gently to suspend them in the solution. Keep the tube vertical in a refrigerator for 10-15 days at - 15°C. The specimens will sink to the bottom of the tube to form a loose pellet during the extraction. c. Washing the Extracted Specimens Remove the glycerol extraction solution from the tube by suction, leaving a pellet of the extracted specimens. Add cold (0-1°C) washing solution I1 to the tube and stir gently to resuspend the extracted specimens in the solution. Centrifuge the tube gently to make a loose pellet of the extracted specimens. Repeat this procedure three times to remove all glycerol and EDTA from the extracted specimens. The washed specimens are to be kept in the washing solution for at least 15 minutes at 0-1°C prior to experimentation. d. Reactivating the Extracted Specimens See Section II,A,4,d. e. Observing and Recording the Reactivated Specimens
The reactivated specimens are observed and photographed through a phasecontrast objective ( x 20). The angle between the ciliary axis and the cell surface at the right anterior edge of the specimen (the “right” is at the observer’s right hand when the side bearing the oral groove is down and the anterior end points away from the observer) is measured on the print of a photographed specimen ( x 400) to determine the degree of ciliary orientation response. D. Triton-Glycerol Extraction of Paramecium As mentioned in the previous section, glycerol destroys the beating mechanism of the ciliary apparatus in Paramecium. Noguchi et al. (1986) performed successive extractions of Paramecium, first by Triton X-100 then by glycerol. Comparison of the reactivated motile activity between Triton-extracted and
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Triton-glycerol-extracted specimens is useful for understanding the functional relationship between the reversal and beating mechanisms in the ciliary motile apparatus.
1. Experimental Solutions Extraction solution I (Triton X-100 extraction solution): This solution is the same as the Triton X-100 extraction solution of Naitoh and Kaneko (1972) (see Section II,A,3). Extraction solution I1 (glycerol extraction solution): 50 mM KCl + 2 mM EDTA + 10 mM Tris-maleate (pH 7.0) + 30% (by volume) glycerol. Washing solution I (for the first Triton-extracted specimens): 50 m M KCl + 2 mM EDTA + 10 mM Tris-rnaleate (pH 7.0). Washing solution I1 (for the second glycerol-extracted specimens): 50 mM KCl + 10 mM Tris-maleate (pH 7.0) + 30% (by volume) glycerol. Test solutions: Basic components of the test solutions are the same as those of washing solution 11. Chemicals to be tested are added to this solution.
2. Experimental Procedures a. Colfecting and Washing the Specimens See Section II,A,4,a. b. Extracting and Washing of Specimens with Triton The procedures are the same as those for the Triton X-100 extraction described in Sections II,A,4,b and c. The extracted and washed specimens are then kept in cold (0-l°C) washing solution I for 15 minutes. d. Extracting the Specimens with Glycerol Centrifuge the washed Triton-extracted specimens gently to make a loose pellet. Resuspend the specimens in ice-cold extraction solution 11. Keep them in an ice bath (OOC) for 1 hour. Then centrifuge them gently to make a loose pellet. e. Washing the Twice-Extracted Specimens Resuspend the extracted specimens in ice-cold washing solution 11. Then centrifuge them gently to make a loose pellet. Repeat this procedure three times to remove EDTA from the extracted specimens. Keep the extracted specimens immersed in the ice-cold washing solution for at least 1 hour prior to experimentation.
f. Reactivating and Observing the Extracted Specimens See Section II,A,4,e.
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E. Modified Triton-Glycerol Extraction of Paramecium To examine the effects of externally applied macromolecules, such as enzymes, antibodies, and polynucleotides, on the motility machinery of the cilia, the membrane of the extracted specimens should be permeable to these molecules. The membrane of Triton X- 100-extracted Paramecium obtained according to the extraction procedures described in the previous sections is not permeable to these macromolecules. In fact, the external application of immunoglobulin causes osmotic shrinkage of the extracted Paramecium. Noguchi (1987) successfully extracted Paramecium with a solution having a higher (1% instead of 0.01%) concentration of Triton X-100. He then examined the effects of externally applied trypsin (trypsin digestion) on the ATPreactivated motile activity of the extracted specimens. Highly permeabilized specimens can be used for immunocytological experimentation. In some cases the effects of an antibody to a specific molecule on the ciliary motile activity can be examined by a simple external application of the antibody without the need for intracellular microinjection (Adoutte et al., 1991, Peranen et al., 1993).
1. Experimental Solutions Washing solution (for both cultured specimens and Triton X- 100-extracted specimens): 50 mM KC1 2 mM EDTA + 10 mM Tris-maleate (pH 7.0) (final concentration). Extraction solution I (Triton X-100 extraction solution): 20 mM KC1 + 10 mM EDTA 10 mM Tris-maleate (pH 7.0) 1% (by volume) Triton x-100. Extraction solution I1 and equilibration solution (glycerol extraction solution): 50 mM KC1 10 mM glycerol 30% (by volume) glycerol. Test solutions: Basic components of the test solutions are 50 mM KC1, 1 mM MgCl,, 1 mM ATP, 10 mM Tris-maleate (pH 7.0), and 30% (by volume) glycerol. Chemicals to be tested are to be added to the basic components.
+
+
+
+
+
2. Experimental Procedures a. Collecting and Washing the Specimens
See Section II,A,4,a.
b. Extracting the Specimens with Triton-Containing Solution Introduce ca. 5 ml of ice-cold extraction solution I into a centrifuge tube containing a loose pellet of washed specimens and resuspend them in the solution for 2 minutes. Centrifuge the specimens gently to make a loose pellet. Remove the extraction solution by suction.
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c. Washing the Extracted Specimens
Introduce ca. 5 ml of ice-cold washing solution into the centrifuge tube to resuspend the extracted specimens. Centrifuge the specimens gently to make a loose pellet of the specimens. Remove the washing solution. Repeat this procedure three times to remove Triton from the specimens. d. Extracting and Equilibrating in Glycerol-Containing Solution
Introduce ca. 5 ml of ice-cold extraction solution I1 into the tube to resuspend the washed extracted specimens in the solution. Keep the specimens in the solution more than 30 minutes. The centrifuge the specimens gently to make a loose pellet. Remove the solution from the tube. Keep the specimens bathed in a minute amount of the equilibration solution in an ice bath. e. Reactivating and obseruating the specimens
See Section II,A,4,e.
F. Isolation of Ciliated Cortical Sheets from Triton-Extracted Paramecium Cilia of Paramecium beat in three dimensions (Machemer, 1972; Naitoh and Sugino, 1984). To determine the exact direction of the effective power stroke or the pointing direction of cilia, it is desirable to look down on the ciliated surface. The profile view of cilia gives only approximate values for these directions. A ciliated cortical sheet isolated from Triton X-100-extracted Paramecium is particularly suitable for observing and recording the top view of cilia. The quality of microscopic images of cilia is far better in the cortical sheet than when viewed through the whole cell, because the cortical sheet is far thinner so that optical disturbance is far less. Thus Noguchi et al. (1991) developed a procedure to isolate ciliated cortical sheets from Triton-extracted Paramecium. Glycerol, which was used in their original experimental solutions, can be eliminated if required for your experimental purposes (Noguchi, 1987; Okamoto and Nakaoka, 1994a,b).
1. Experimental Solutions Washing solution I (both for cultured and extracted specimens): 50 mM 10 mM Tris-maleate (pH 7.0) (in final concenKCI + 2 mM EDTA tration). 2. Extraction solution: 20 mM KCI + 10 mM EDTA + 10 mM Tris-maleate (pH 7.0) + 1% (by volume) Triton X-100. KCL-glycerol solution: 50 mM KCI 10 mMTris-maleate (pH 7.0) + 30% (by volume) glycerol. Test solutions: Basic components of the test solutions are 50 mM KCI, 1 mM MgCl,, 1 mM ATP, 10 mM Tris-maleate (pH 7.0), and 30% (by
+
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volume) glycerol. Free Ca2+concentration is controlled by a Ca-EGTA buffer with 1 mM EGTA.
2. Procedures a. Collecting and Washing the Cultured Specimens
See Section II,A,4,a.
6. Extracting the Specimens
Suspend washed and concentrated specimens in the ice-cold extraction solution for 2 minutes. Centrifuge them gently to make a loose pellet. Remove the extraction solution by suction. c. Washing the Extracted Specimens
Resuspend the extracted specimens in ice-cold washing solution. Centrifuge them gently to form a loose pellet. Repeat this procedure three times to remove Triton from the extracted specimens. d. Fragmenting the Washed Extracted Specimens
Pipet a suspension of the extracted specimens in and out several times to fragment the specimens. The inner diameter of the pipet should be ca. 50 pm. A sharp broken edge of the orifice of the pipet is effective in fragmenting the specimens. Keep fragmented specimens suspended in ice-cold equilibration solution until used. e. Reactivating and Observing the Cortical Sheets
The following procedures are carried out at a room temperature of 23-25°C. Pipet about 20 pl of the fragment suspension on a glass slide. Place a coverslip with a small amount of vaseline on two opposite edges on the suspension to keep the fragments in a thin space between the coverslip and the glass slide. Adjust the thickness of the space by controlling the amount of vaseline and the force used to press the coverslip onto the glass slide. Then gently pipet the equilibration solution into the space from its vaselinefree side, and remove excess solution from the opposite side by placing the edge of a piece of filter paper on it. During this perfusion procedure, some of the fragments will attach with their cellular side flat against the glass slide (or coverslip). The ciliated cortical sheets thus obtained are ready for perfusion of a test solution. Images of cilia on the cortical sheet are observed and recorded as described in a previous section (see Section II,A,4,e).
Acknowledgments I thank Dr. R. D. Allen and Dr. A. K. Fok for their critical reading of the manuscript which was prepared in their laboratory with the support of National Science Foundation grants MCB9017455 and MCB9206097.
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References Adoutte, A., Delgado, P., Fleury, A., Levilliens, N., Laine, M. C., Marty, M. C., Boisvieux-Ulrich, E., and Sandoz, D. (1991). Microtubule diversity in ciliated cells: evidence for its generation by post-translational modification in the axonemes of Paramecium and quail oviduct cells. Biol. Cell 71, 227-245. Bonini, N. M., and Nelson, D. L. (1988). Differential regulation of Paramecium ciliary motility by CAMP and cGMP. J. Cell Biol. 106, 1615-1623. Bonini, N. M., Gustin, M. C., and Nelson, D. L. (1986).Regulation of ciliary motility by membrane potential in Paramecium: A role for cyclic AMP. Cell Motil. Cytoskel. 6, 256-272. Eckert, R. (1972). Bioelectric control of ciliary activity. Science 176,473-481. Epstein, M.,and Eckert, R. (1973). Membrane control of ciliary activity in the protozoan Euplotes. J . Exp. Biol. 58, 437-462. Gibbons, B. H., and Gibbons, 1. R. (1972). Flagellar movement and adenosinetriphosphate activity in sea urchin sperm extracted with Triton X-100.J . Cell Biol. 54, 75-97. Gibbons, I. R., Evans, J. A., and Gibbons, B. H. (1982). Acetate anions stabilize the latency of dynein 1 ATPase and increase the velocity of tubule sliding in reactivated sperm flagella. Cell Moril. l(Suppl.), 18I- 184. Goodenough, V. W. (1983). Motile detergent-extracted cells of Tetrahymena and Chlamydomonas. J . Cell Biol. 96, 1610-1621. Hamasaki, T.,and Naitoh, Y. (1985). Localization of calcium sensitive reversal mechanism in a cilium of Paramecium. Proc. Jpn Acad. 61B, 140-143. Hamasaki, T., Barkalow, K., Richmond, J., and Satir, P. (1991).Cyclic AMP-stimulatedphosphorylation of an axonemal polypeptide that copurifies with the 22s dynein arm regulates microtubular translocation velocity and swimming speed in Paramecium. Proc. Natl. Acad. Sci. U.S.A. 88, 79 18-7922. Hamasaki, T., Murtaugh, T. J., Satir, B. H., and Satir, P. (1989). In vitro phosphorylation of Paramecium axonemes and permeabilized cells. Cell Motil. Cytoskel. U ,1-1 1. Hoffman-Berling, H. (1955). Geiselmodelle und Adenosintriphosphat. Biochim. Biophys. Acta 16, 146- 154. Kung, C., and Naitoh, Y. (1973). Calcium-induced ciliary reversal in the extracted models of “pawn”, a behavioral mutant of Paramecium. Science 179, 195-196. Lieberman, S.J., Hamasaki, T., and Satir, P. (1988). Ultrastructure and motion analysis of permeabilized Paramecium capable of motility and regulation of motility. Cell Motil. Cytoskel. 9, 73-84. Machemer, H. (1972). Ciliary activity and the origin or metachrony in Paramecium: Effects of increased viscosity. J. Exp. Biol. 65,427-448. Majima, T., Hamasaki, T., and Arai, T. (1986). Increase in cellular cyclic GMP level by potassium stimulation and its relation to ciliary orientation in Paramecium. Experientia 42, 62-64. Naitoh, Y. (1966). Reversal response elicited in nonbeating cilia of Paramecium by membrane depolarization. Science 154, 660-662. Naitoh, Y. (1969). Control of the orientation of cilia by adenosinetriphosphate, calcium and zinc in glycerol-extracted Paramecium caudatum. J. Gen. Physiol. 53, 517-529. Naitoh, Y. (1981). Membrane currents in voltage-clamped Paramecium caudatum. In “Nerve Membrane Biochemistry and Function of Channel Protein” ( G . Matsumoto and M. Kotani, eds.), pp. 113-117. University of Tokyo Press, Tokyo. Naitoh, Y. (1982). Protozoa. In “Electric Conduction and Behaviour in ‘Simple’ Invertebrates” (G. A. B. Shelton, ed.), pp. 1-48, Clarendon Press, Oxford. Naitoh, Y., and Eckert, R. (1969). Ionic mechanisms controlling behavioral responses of Paramecium to mechanical stimulation. Science 164,963-965. Naitoh, Y.,and Kaneko, H. (1972). Reactivated Triton-extracted models of Paramecium: modification of ciliary movement by calcium ions. Science 172, 523-524. Naitoh, Y ., and Kaneko, H. (1973).Control of ciliary activity by adenosinetriphosphateand divalent cation in Triton-extracted models of Paramecium caudatum. J. Exp. Biol. 58, 657-676.
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Reactivation of Extracted Paramecium Models Yutaka Naitoh Pacific Biomedical Research Center and Department of Microbiology University of Hawaii at Manoa Honolulu. Hawaii 96822
I. Introduction 11. Methods
A. Triton X-100 Extraction of Paramecium B. Modified Triton X-100 Extraction of Paramecium C. Glycerol Extraction of Paramecium D. Triton-Glycerol Extraction of Paramecium E. Modified Triton-Glycerol Extraction of Paramecium F. Isolation of Ciliated Cortical Sheets from Triton-Extracted Paramecium References
I. Introduction The swimming behavior of Paramecium is dependent mostly on its ciliary motile activity. Ciliary motile activity is regulated by membrane electrogenesis (Naitoh and Eckert, 1969; Eckert, 1972, Naitoh, 1982). A depolarizing receptor potential evoked by stimulation of the front end of Paramecium electrotonically spreads over the entire cell membrane to activate (open) depolarization-sensitiveCa2+channels in the ciliary membrane. Activation of the channels causes regenerative entry of Ca2+ ions into the cilia, producing a sudden increase in the intraciliary Ca2+concentration and an action potential. The Ca2+increase activates a mechanism for reversing the direction of the effective power stroke of cilia to cause backward swimmingof Paramecium. Paramecium thereby avoids a noxious stimulus at its front. METHODS IN CELL BIOLOGY,VOL. 47 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved
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A hyperpolarizing receptor potential evoked by stimulation of the posterior end of Paramecium also spreads electrotonically over the entire cell membrane. Hyperpolarization of the membrane brings about an increase in the intracellular CAMP concentration, which activates the ciliary beating mechanism to cause an increase in the frequency of ciliary beating in the normal direction (Nakaoka et al., 1984; Majima et al., 1986; Bonini et al., 1986). This brings about rapid forward swimming of the specimen. Paramecium thereby escapes from a noxious stimulus at its rear. To understand the mechanism by which ciliary motile activity is regulated by these chemical messengers, it is important to examine the direct effects of the chemicals on the motile activity of the ciliary apparatus. An external application of these chemicals does not, however, necessarily increase the intracellular concentrations of the chemicals, as the cell membrane is poorly permeable to these chemicals. Moreover, an external application of Ca2+ions will even produce a decrease in the intracellular Ca2+concentration in Paramecium, because it deactivates Ca2+channels so that Ca2+influx decreases (Naitoh, 1981). Therefore, permeabilization of the cell membrane is one method essential for examining the direct effects of the chemicals. In a permeabilized cell, functions of the membrane as a signal transducer and a diffusion barrier are disrupted, while the motor function of the ciliary apparatus is kept normal. The idea of permeabilization of the cell originated from a classic work on the glycerinated muscle fiber by Szent-Gyorgyi (1947). Hoffman-Berling (1955) applied Szent-Gyorgyi’s technique to protozoan cells to make “ciliary models.” Gibbons and Gibbons (1972) first introduced a nonionic detergent, Triton X100, to obtain permeabilized seaurchin spermatozoa so good that almost 100% of them showed ATP-Mg2+-reactivated flagellar motion. It should be noted that a detergent destroys not only the membrane but also the ciliary apparatus. The concentration of the detergent, the time of exposure of the cells to the detergent, the ambient temperature, and the ionic conditions all influence the quality of the extracted models. Tolerance to a given detergent differs from specimen to specimen depending on their culture condition, cell size, species, and strains. Although Triton X100 is the most popular detergent for permeabilization of Paramecium, it is worthwhile examining other detergents as well, such as Nonidet P-40 (Goodenough, 1983) and saponin (Seravin, 1961). In addition to ATP, Mg2+, and KCl, other chemicals in the reactivation solution are important to keep the reactivated ciliary activity stable. Acetate ions, instead of C1- (Gibbons et al., 1982), and polyethylene glycol (Goodenough, 1983) have been used to stabilize the ciliary activity. It is important to determine your own conditions for permeabilization adequate for your experimental purposes. Therefore, the recipes given in the following sections are subject to your own modification.
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11. Methods A. Triton X-100 Extraction of Paramecium
This standard procedure of Naitoh and Kaneko (1972) for Triton X-100 extraction of Paramecium caudatum has been employed, with minor modifications, by many authors for other species of Paramecium (P.tetraurelia: Kung and Naitoh, 1973; Bonini et al., 1986; Lieberman et al., 1988; P . multimicronucleaturn: Okamoto and Nakaoka, 1994) and other genera of ciliates, including Tetrahymena (Takahashi et al., 1980; Goodenough, 1983) and Euplotes (Epstein and Eckert, 1973).
1. Culture of Specimens Specimens obtained from any kind of culture (bacterized hay, rice straw, wheat straw, lettuce, or milk infusion) can be used. Crude cultures, such as bacterized hay infusion made over a layer of field soil in a ceramic bowl, are preferable to the more sophisticated axenic cultures to obtain good extracted specimens. Such specimens show coordinated and long-lasting ciliary movement when reactivated. Axenic culture, however, is essential for more critical biochemical examinations of the extracted specimens.
2. Stock Solutions 0.1 M KCI 0.1 M CaCI, 0.1 M MgCl, 0.1 M EDTA (neutralized by KOH) 0.1 M EGTA (neutralized by KOH) 0.1 M Tris-maleate pH buffer (PH 7.0)
0.1 M Tris-HC1 pH buffer (pH 7.4) 0.1 M ATP (neutralized by KOH, to be stored in a freezer) 0.1 M KOH 0.1 M HC1 1% (by volume) Triton X-100
3. Experimental Solutions Washing solution I (for living specimens from culture): 2 mM CaCI, + 1 mM Tris-HC1 (pH 7.2) (in final concentration). Mix 10 ml of 0.1 M CaCI,, with 5 ml of 0.1 M Tris-HC1 and add H,O to make 500 ml of solution. Extraction solution: 20 mM KCI + 10 mM ethylenediaminetetraacetic acid (EDTA) + 10 mM Tris-maleate (pH 7.0) + 0.01% (by volume) Triton X-100. Mix 100 ml of 0.1 M KC1, 50 ml of 0.1 M EDTA, 50 ml of 0.1 M Tris-maleate (pH 7.0), and 5 ml of 1% Triton X-100, and add H,O to make 500 ml of solution.
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Washing solution I1 (for extracted specimens): 50 mM KCl + 10 mM Tris-maleate (pH 7.0). Mix 250 ml of 0.1 M KCl with 50 ml of 0.1 MTris-maleate and add H 2 0 to make 500 ml of solution. Test solutions: Basic components of the test solutions are the same as those of the washing solution 11. Chemicals to be tested are added to the solution. Two representative test solutions are:
1. Standard reactivation solution: 50 mM KC1 + 4 mM MgCl, + 4 mM ATP + 3 mM ethylene glycol bis (B-aminoethyl ether)-N,N-tetraacetic acid (EGTA) + 10 mM Tris-maleate (pH 7.0). Mix 5 ml of 0.1 M KCl, 0.4 ml of 0.1 M MgCl,, 0.4 ml of 0.1 M ATP, 0.3 ml of 0.1 M EGTA, and 1 ml of 0.1 M Tris-maleate and add H,O to make 10 ml of solution. The extracted specimens swim forward in this solution. 2. Ca series reactivation solutions: Add an amount of 0.1 M CaCl, to be determined by the experiment to the standard reactivation solution. The concentration ratio of Ca2+to EGTA determines the free Ca2+concentration in the solution (Ca2+buffer; Portzehl et ul., 1964;Noguchi et ul., 1986).The extracted specimens swim backward when the free Ca2+concentration in the solution is above M.
4. Experimental Procedures a. Collecting and Washing the Specimens
Introduce about 20 ml of culture medium containing specimens of Purumecium into a long-necked flask (such as a 50-ml volumetric flask). Fill the flask with washing solution I. Keep the flask still on a table for ca. 4 minutes. Specimens will gather at the neck portion of the flask due to their negative geotaxis. Pipet the specimens into another long-necked flask, then fill the flask with the washing solution. The specimens again gather in the neck portion. Repeat this procedure three times to dilute the culture medium. Pipet the concentrated specimens in the neck portion into a centrifuge tube. Keep the tube in an ice bath for 3 minutes. Centrifuge the tube gently to make a loose pellet of the specimens. Remove the supernatant by suction. 6. Extracting the Specimens Introduce ca. 5 ml of cold (0-1°C) extraction solution into the tube to suspend the washed specimens in the solution. Keep the tube vertical in an ice bath for ca. 30 minutes. Specimens gradually lose their mobility in the extraction solution and sink toward the bottom of the tube. Remove the extraction solution by suction, leaving the extracted specimens at the bottom. c. Washing the Extracted Specimens
Introduce ca. 5 ml of cold (0-l°C) washing solution I1 into the tube to suspend the extracted specimens in the solution. Centrifuge the tube very gently to
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make a loose pellet of the specimens. Remove the supernatant by suction. Repeat this procedure three times to wash the Triton X-100and EDTA away from specimens. Keep the extracted specimens cooled in an ice bath, in a minute amount of the washing solution. d. Reactivating the Extracted Specimens Pipet about 1 pl of washing solution containing hundreds of extracted specimens into ca. 1 ml of a test solution on a depression slide at room temperature (ca. 24°C). Stir the mixture gently. A few seconds after this mixing, the extracted specimens will begin to swim due to the reactivation of the ciliary movement, providing the test solution contains an adequate amount of ATP and Mg2+. e. Observing and Recording Reactivated Specimens Pipet about 100 pl of test solution containing scores of reactivated specimens onto a glass slide to make a droplet. Put several small pieces of crushed coverslip into the droplet so that a thin space is made when a coverslip is put on the droplet. The extracted specimens will swim freely in the thin space between the glass slide and the coverslip. Put white vaseline around the edge of the coverslip to prevent evaporation of the test solution. The specimens are now ready for observation and recording of their motile activity. Swimming Paths of the Reactivated Specimens. Under dark-field illumination, swimming specimens are easily observed through a conventional binocular dissecting microscope with a final magnification of x 10- x 20. Movement of the specimens can be recorded with a conventional video recorder. The swimming path of a specimen is then determined by frame-by-frame analysis of displayed images of the specimen. Various kinds of computerized analyzers are available for this type of analysis (e.g., see Chapter 40 in this volume). The swimming path of a specimen can be photographed as a curved spiral line on a still film when the exposure time is extended to 1-5 seconds. Swimming direction and swimming velocity can be determined from the swimming path. Ciliary Motile Activity. A conventional phase-contrast, interferencecontrast, or dark-field microscope with a final magnification of X 200- X 400 is essential for observing and recording the reactivation of the cilia. High-speed video recording of the cilia is essential to determine the beat frequency, the direction of the effective power stroke, and the beating form of an individual cilium (see Chapter 34 in this volume). The computerized image contrastenhancement technique is useful for obtaining a good image of a cilium (see Chapter 38 in this volume). The beat frequency of reactivated cilia of an extracted specimen can be determined from the frequency of change in light intensity of a selected small area of a magnified image of the specimen due to the passage of metachronal waves. This is accomplished by placing a photodiode or a photomultiplier in the path of the image (Naitoh and Kaneko, 1973). The relative force exerted by the reactivated cilia on a given specimen can
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be estimated from the swimming path of the specimen (Naitoh and Sugino, 1984). The direction of the force corresponds to the mean direction of the effective power strokes, and the amount of force is proportional to the mean beat frequency of all the cilia. Ciliary metachronal waves can be observed as darker (or brighter) lines moving on the ciliary surface or as waves moving along the margin of a reactivated specimen. The metachronal wave lines are easily photographed on film. The direction of the effective power stroke can be determined from the metachronal wave line, as the angle between the metachronal wave line and the direction of the effective power stroke is always 90"(Machemer, 1972). The direction in which nonbeating cilia point, which usually corresponds to the direction of the effective power stroke, can be determined from a photograph of an extracted specimen.
B. Modified Triton X-100 Extraction of Paramecium Nakaoka and Toyotama (1979) added Mg2+ ions to the Triton X-100containing extraction solution. Specimens extracted with this solution showed higher sensitivity to Ca2+in modifying beat frequency in Mg2+-ATP-reactivated cilia than those extracted with a solution without Mg2+.The maximum beat frequency under optimum reactivation conditions was higher in the Mg2+containing extraction solution and close to the maximum beat frequency of living specimens. Formation of metachronal waves on the reactivated ciliary surface was also enhanced (Nakaoka et al., 1984; Nakaoka and Ooi, 1985). The presence of Mg2+ in the extraction solution maintains the ciliary motile mechanisms of Paramecium in a closer-to-normal condition than when Mg2+ is absent during the Triton X-100 treatment.
1. Experimental Solutions Extraction solution: 20 mM KCl + 5 mM MgCl, + 5 mM EGTA + 0.008% (by volume) Triton X-100 + 10 m M Tris-maleate buffer (pH 7.0) (in final concentration). Washing solution I: 20 mM KCl + 5 mM MgC1, + 3 mM EGTA + 10 mM Tris-maleate buffer (pH 7.0). Washing solution 11: 20 mM KCl + 5 mM MgCl, + mM Tris-maleate buffer (pH 7.0). Reactivation solutions: 10 mM KC1 + 8 mM MgCl, + 4 mM ATP + 1 mM EGTA + an amount of CaC1, needed to provide the appropriate free Ca2+ concentration required for the experiment + 10 mM Tris-maleate buffer (pH 7.0).
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2. Experimental Procedures a. Collecting and Washing Cultured Specimens See Section II,A,4,a.
b. Extracting the Specimens Suspend the washed specimens in the extraction solution in a centrifuge tube for 10-12 minutes at room temperature (25°C). Remove the extraction solution by suction, leaving the extracted specimens at the bottom of the tube. c. Washing the Extracted Specimens Introduce cold (OOC) washing solution I into the tube to suspend the extracted specimens in the solution. Keep the tube vertical in an ice bath for 15 minutes. The extracted specimens will sink toward the bottom of the tube. Remove the washing solution by suction, leaving the specimens at the bottom of the tube. Introduce cold (OOC) washing solution I1 into the tube to suspend the specimens. Keep the tube in an ice bath. The specimens sink toward the bottom in 15 minutes. Washed specimens thus obtained are ready for experimentation. d. Reactivating the Extracted Specimens Pipet a concentrated suspension of the extracted specimens into ca. 1 ml of a reactivation solution at room temperature (20-22°C). Stir the mixture gently for a few seconds. Observations and measurements are made 3-20 minutes after mixing at room temperature.
C. Glycerol Extraction of Paramecium Glycerol-extracted Paramecium models do not show ciliary beating in the presence of ATP and Mg2+; however, glycerol extraction supports a change in the direction in which nonbeating cilia point on administration of ATP and Ca2+. That is, cilia swing once to change their pointing direction (ciliary reorientation response). The reorientation response in nonbeating cilia corresponds to a change or reversal of the direction of the effective power stroke (ciliary reversal) in beating cilia (Naitoh, 1966). Glycerol, therefore, specifically destroys the beating mechanism in the ciliary motile apparatus.
1. Experimental Solutions
Washing solution I (for specimens from culture): 2 mM CaCl, + 1 mM Tris-HC1 (pH. 7.2) (in final concentration). 10 mM Tris-HC1 Extraction solution: 50 mM KC1 10 mM EDTA (pH 7.4) + 50% (by volume) glycerol.
+
+
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Washing solution I1 (for extracted specimens): 50 mM KCl + 10 mM Tris-HC1 (PH 7.4). Test solutions: Basic components of a test solution are the same as those of the washing solution I1 except for pH (pH is 9.0 instead of 7.4 in the washing solution). Chemicals to be tested are added to the basic solution.
2. Experimental Procedures a. Collecting and Washing the Cultured Specimens See Section II,A,4,a.
b. Extracting the Specimens Introduce ca. 5 ml of cold (0-1°C) extraction solution into the centrifuge tube containing a pellet of washed specimens, and stir gently to suspend them in the solution. Keep the tube vertical in a refrigerator for 10-15 days at - 15°C. The specimens will sink to the bottom of the tube to form a loose pellet during the extraction. c. Washing the Extracted Specimens Remove the glycerol extraction solution from the tube by suction, leaving a pellet of the extracted specimens. Add cold (0-1°C) washing solution I1 to the tube and stir gently to resuspend the extracted specimens in the solution. Centrifuge the tube gently to make a loose pellet of the extracted specimens. Repeat this procedure three times to remove all glycerol and EDTA from the extracted specimens. The washed specimens are to be kept in the washing solution for at least 15 minutes at 0-1°C prior to experimentation. d. Reactivating the Extracted Specimens See Section II,A,4,d. e. Observing and Recording the Reactivated Specimens
The reactivated specimens are observed and photographed through a phasecontrast objective ( x 20). The angle between the ciliary axis and the cell surface at the right anterior edge of the specimen (the “right” is at the observer’s right hand when the side bearing the oral groove is down and the anterior end points away from the observer) is measured on the print of a photographed specimen ( x 400) to determine the degree of ciliary orientation response. D. Triton-Glycerol Extraction of Paramecium As mentioned in the previous section, glycerol destroys the beating mechanism of the ciliary apparatus in Paramecium. Noguchi et al. (1986) performed successive extractions of Paramecium, first by Triton X-100 then by glycerol. Comparison of the reactivated motile activity between Triton-extracted and
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Triton-glycerol-extracted specimens is useful for understanding the functional relationship between the reversal and beating mechanisms in the ciliary motile apparatus.
1. Experimental Solutions Extraction solution I (Triton X-100 extraction solution): This solution is the same as the Triton X-100 extraction solution of Naitoh and Kaneko (1972) (see Section II,A,3). Extraction solution I1 (glycerol extraction solution): 50 mM KCl + 2 mM EDTA + 10 mM Tris-maleate (pH 7.0) + 30% (by volume) glycerol. Washing solution I (for the first Triton-extracted specimens): 50 m M KCl + 2 mM EDTA + 10 mM Tris-rnaleate (pH 7.0). Washing solution I1 (for the second glycerol-extracted specimens): 50 mM KCl + 10 mM Tris-maleate (pH 7.0) + 30% (by volume) glycerol. Test solutions: Basic components of the test solutions are the same as those of washing solution 11. Chemicals to be tested are added to this solution.
2. Experimental Procedures a. Colfecting and Washing the Specimens See Section II,A,4,a. b. Extracting and Washing of Specimens with Triton The procedures are the same as those for the Triton X-100 extraction described in Sections II,A,4,b and c. The extracted and washed specimens are then kept in cold (0-l°C) washing solution I for 15 minutes. d. Extracting the Specimens with Glycerol Centrifuge the washed Triton-extracted specimens gently to make a loose pellet. Resuspend the specimens in ice-cold extraction solution 11. Keep them in an ice bath (OOC) for 1 hour. Then centrifuge them gently to make a loose pellet. e. Washing the Twice-Extracted Specimens Resuspend the extracted specimens in ice-cold washing solution 11. Then centrifuge them gently to make a loose pellet. Repeat this procedure three times to remove EDTA from the extracted specimens. Keep the extracted specimens immersed in the ice-cold washing solution for at least 1 hour prior to experimentation.
f. Reactivating and Observing the Extracted Specimens See Section II,A,4,e.
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E. Modified Triton-Glycerol Extraction of Paramecium To examine the effects of externally applied macromolecules, such as enzymes, antibodies, and polynucleotides, on the motility machinery of the cilia, the membrane of the extracted specimens should be permeable to these molecules. The membrane of Triton X- 100-extracted Paramecium obtained according to the extraction procedures described in the previous sections is not permeable to these macromolecules. In fact, the external application of immunoglobulin causes osmotic shrinkage of the extracted Paramecium. Noguchi (1987) successfully extracted Paramecium with a solution having a higher (1% instead of 0.01%) concentration of Triton X-100. He then examined the effects of externally applied trypsin (trypsin digestion) on the ATPreactivated motile activity of the extracted specimens. Highly permeabilized specimens can be used for immunocytological experimentation. In some cases the effects of an antibody to a specific molecule on the ciliary motile activity can be examined by a simple external application of the antibody without the need for intracellular microinjection (Adoutte et al., 1991, Peranen et al., 1993).
1. Experimental Solutions Washing solution (for both cultured specimens and Triton X- 100-extracted specimens): 50 mM KC1 2 mM EDTA + 10 mM Tris-maleate (pH 7.0) (final concentration). Extraction solution I (Triton X-100 extraction solution): 20 mM KC1 + 10 mM EDTA 10 mM Tris-maleate (pH 7.0) 1% (by volume) Triton x-100. Extraction solution I1 and equilibration solution (glycerol extraction solution): 50 mM KC1 10 mM glycerol 30% (by volume) glycerol. Test solutions: Basic components of the test solutions are 50 mM KC1, 1 mM MgCl,, 1 mM ATP, 10 mM Tris-maleate (pH 7.0), and 30% (by volume) glycerol. Chemicals to be tested are to be added to the basic components.
+
+
+
+
+
2. Experimental Procedures a. Collecting and Washing the Specimens
See Section II,A,4,a.
b. Extracting the Specimens with Triton-Containing Solution Introduce ca. 5 ml of ice-cold extraction solution I into a centrifuge tube containing a loose pellet of washed specimens and resuspend them in the solution for 2 minutes. Centrifuge the specimens gently to make a loose pellet. Remove the extraction solution by suction.
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c. Washing the Extracted Specimens
Introduce ca. 5 ml of ice-cold washing solution into the centrifuge tube to resuspend the extracted specimens. Centrifuge the specimens gently to make a loose pellet of the specimens. Remove the washing solution. Repeat this procedure three times to remove Triton from the specimens. d. Extracting and Equilibrating in Glycerol-Containing Solution
Introduce ca. 5 ml of ice-cold extraction solution I1 into the tube to resuspend the washed extracted specimens in the solution. Keep the specimens in the solution more than 30 minutes. The centrifuge the specimens gently to make a loose pellet. Remove the solution from the tube. Keep the specimens bathed in a minute amount of the equilibration solution in an ice bath. e. Reactivating and obseruating the specimens
See Section II,A,4,e.
F. Isolation of Ciliated Cortical Sheets from Triton-Extracted Paramecium Cilia of Paramecium beat in three dimensions (Machemer, 1972; Naitoh and Sugino, 1984). To determine the exact direction of the effective power stroke or the pointing direction of cilia, it is desirable to look down on the ciliated surface. The profile view of cilia gives only approximate values for these directions. A ciliated cortical sheet isolated from Triton X-100-extracted Paramecium is particularly suitable for observing and recording the top view of cilia. The quality of microscopic images of cilia is far better in the cortical sheet than when viewed through the whole cell, because the cortical sheet is far thinner so that optical disturbance is far less. Thus Noguchi et al. (1991) developed a procedure to isolate ciliated cortical sheets from Triton-extracted Paramecium. Glycerol, which was used in their original experimental solutions, can be eliminated if required for your experimental purposes (Noguchi, 1987; Okamoto and Nakaoka, 1994a,b).
1. Experimental Solutions Washing solution I (both for cultured and extracted specimens): 50 mM 10 mM Tris-maleate (pH 7.0) (in final concenKCI + 2 mM EDTA tration). 2. Extraction solution: 20 mM KCI + 10 mM EDTA + 10 mM Tris-maleate (pH 7.0) + 1% (by volume) Triton X-100. KCL-glycerol solution: 50 mM KCI 10 mMTris-maleate (pH 7.0) + 30% (by volume) glycerol. Test solutions: Basic components of the test solutions are 50 mM KCI, 1 mM MgCl,, 1 mM ATP, 10 mM Tris-maleate (pH 7.0), and 30% (by
+
+
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volume) glycerol. Free Ca2+concentration is controlled by a Ca-EGTA buffer with 1 mM EGTA.
2. Procedures a. Collecting and Washing the Cultured Specimens
See Section II,A,4,a.
6. Extracting the Specimens
Suspend washed and concentrated specimens in the ice-cold extraction solution for 2 minutes. Centrifuge them gently to make a loose pellet. Remove the extraction solution by suction. c. Washing the Extracted Specimens
Resuspend the extracted specimens in ice-cold washing solution. Centrifuge them gently to form a loose pellet. Repeat this procedure three times to remove Triton from the extracted specimens. d. Fragmenting the Washed Extracted Specimens
Pipet a suspension of the extracted specimens in and out several times to fragment the specimens. The inner diameter of the pipet should be ca. 50 pm. A sharp broken edge of the orifice of the pipet is effective in fragmenting the specimens. Keep fragmented specimens suspended in ice-cold equilibration solution until used. e. Reactivating and Observing the Cortical Sheets
The following procedures are carried out at a room temperature of 23-25°C. Pipet about 20 pl of the fragment suspension on a glass slide. Place a coverslip with a small amount of vaseline on two opposite edges on the suspension to keep the fragments in a thin space between the coverslip and the glass slide. Adjust the thickness of the space by controlling the amount of vaseline and the force used to press the coverslip onto the glass slide. Then gently pipet the equilibration solution into the space from its vaselinefree side, and remove excess solution from the opposite side by placing the edge of a piece of filter paper on it. During this perfusion procedure, some of the fragments will attach with their cellular side flat against the glass slide (or coverslip). The ciliated cortical sheets thus obtained are ready for perfusion of a test solution. Images of cilia on the cortical sheet are observed and recorded as described in a previous section (see Section II,A,4,e).
Acknowledgments I thank Dr. R. D. Allen and Dr. A. K. Fok for their critical reading of the manuscript which was prepared in their laboratory with the support of National Science Foundation grants MCB9017455 and MCB9206097.
31. Reactivation of Extracted Paramecium
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