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J. PhyS. Chem. 1981, 85,3295-3299
Physicochemical Studies of Chemoreception in Tetrahymena pyriformis by Use of Fluorescence Probes Hiroshl Tanabe, Naokl Kamo, and Yonosuke Kobatake" Facuw of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan (Received: May 19, 1981; In Final Form: July 8, 1981)
Changes of the surface potential, membrane potential, and fluidity of Tetrahymena pyriformis membrane associated with chemoreception were examined by use of fluorescence probes. These changes in membrane properties were compared to chemotactic responses to chemical stimuli. The initial process of chemoreception in T. pyriformis is discussed in terms of physical chemistry.
Introduction Recent studies on chemotaxis of bacteria by biochemical and genetic methods have significantly advanced our knowledge of the molecular processes in chemoreception. Some specific proteins which are responsible for chemoreception have been isolated and identified in bacteria-l Specific binding of chemical stimuli to receptor proteins in a membrane system is considered to be the initial process of chemoreception. Similar notions have been applied in the analysis of hormonal action and synaptic junction in higher vertebrates. A key unanswered question is the following are specific interactions between chemicals and binding sites the sole causes of chemoreception? As a complementary approach to this question, one can try to elucidate the mechanism of chemoreception and -taxis on physicochemicalbases in which the processes of reception, recognition, and transduction are interpreted in terms of a more general molecular interaction between chemical stimuli and membranes. Physicochemicaltechniques such as fluorescence analysis may be extended for use in appropriate living microorganisms without disrupting the cells. In this connection, a ciliated protozoan, Tetrahymena pyriformis, is a suitable system. The present article stresses that the physicochemical concepts are reasonably applicalbe to the interpretations of chemoreception and -taxis in T. pyriformis, and elucidate answeres to the questions: How does the cell or surface membrane perceive molecular interactions such as electrostatic and hydrophobic ones as well as specific binding? How is the sensed information transduced into the motile system to result in taxis in T. pyriformis? The fluorescent probes used here are 8-anilinonaphthalenesulfonate (ANS), 1,6-diphenyl-l,3,5-hexatriene (DPH), and rhodamine 6G (Rh6G). We also examined what information these fluorescent probes provide about simple membranes composed of phospholipid bilayers, i.e., liposomes. Materials and Methods Preparation of Liposomes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were extracted from egg yolk with chloroform-methanol (2:l by volume) and purified by the method of Lea et ala2 For some experiments, lipids extracted from T. pyriformis were used. T. pyriformis was cultured with the method of Bligh and Dyer.3 The lipids obtained were dissolved in chloroform and stored at -20 "C under nitrogen gas until used. Two types of liposomes were employed; multilamella and sonicated single-wall liposomes. The former was used for (1)Adler, J. Annu. Reu. Biochern. 1975,44, 341. (2) Lea, C.H.; Rhodes, D. W.; Stell, R. D. Biochem. J. 1955,60,353. (3) Bligh, E.C.; Dyer, W. J. Can. J. Biochem. Physiol. 1959,37,911.
the electrophoretic experiments (zeta-potential measurement). Lipids dissolved in chloroform were dried under vacuum to deposit a thin film of lipid on the wall of a flask, to which an appropriate salt solution was added. The lipids were dispersed by agitating the flask with a Vortex mixer until all of the lipids had been freed from the wall. When single-wall liposomes were necessary, the dispersed lipids were sonicated for 60 min, and the suspension obtained was centrifuged at 105gfor 20 min. The supernatant was used for the experiments. Fluorescence Measurements. ANS was purchased from Eastman Kodak Co. and purified by the method of Weber and Young: Its fluorescence was excited at 370 nm and monitored at 480 nm with a Hitachi MPF-2A. Rh6G [commercial name rhodamine 6G0, 3,6-bis(ethylamino)-2,7-dimethyl-9-(2'-carbethoxypheny1)xanthenyl chloride], was purchased from Chroma-Gesellschaft Schmit & Co.; its fluorescence was excited at 520 nm and monitored at 550 nm. For the subsequent discussion, the change in the fluorescence intensity, A f , is defined as Af = [ ( f - f o ) / f o l
X
100%
where f and f o are the fluoresence intensities in dye-T. pyriformis suspension in the presence and absence of chemical stimuli, respectively. T. pyriformis was labeled with 1pM DPH, obtained from Tokyo Chemical Industry Co. Its emission was excited at 360 nm and detected at 430 nm through a 390-nm cutoff filter. The degree of fluorescence polarization, P, was defined by
p = (Ill
- 11)/(111 + Il.1
where Ill and I , are the fluorescence intensities when polarizer and analyzer are parallel and perpendicular. Chemotaxis in Tetrahymena p y r i f ~ r m i s . T. ~ pyriformis was grown at 22 "C in a medium containing 2% proteose pepton, 1%yeast extracts, and 0.6% glucose. The cells taken from a 3-day old culture were used for the experiments. They were collected by gentle filtration through filter paper (Whatman No. 3) and washed throughly with control solution (1mM Tris-HC1buffer at pH 7.0). The chemotactic response was measured as follows. An equal number of organisms was suspended in the control and stimulating solutions. The stimulating solution was prepared by dissolving the chemical stimulus in an aliquot of the control solution. Adjacent sides of a shallow vessel were filled with the two solutions. After 25 min, the number or organisms in microphotographs of the stimulating solution ( n J and the control solution (n2)were (4)Weber, G.;Young, L. B. J. Bid. Chem. 1964,239,1415. (5) Tanabe, H.; Kurihara, K.; Kobatake, Y . Biochim. Biophys. Acta 1979,553,369.
0022-365418112085-3295$01.2510 @ 1981 American Chemical Society
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The Journal of Physical Chemistry, Vol. 85, No. 22, 1987
Tanabe et al.
3-
I
t -7 -6 - 5 - 4 -3 -2 -1
log [salt]
0
1
6
8
10
( x ~ ~ - 5 ~ )
Flgure 2. Fluorescence intensity of ANS as a function of total concentration of ANS added to liposomal suspensions.
PE
0 NaCl
0 c 3 0 0
4
KCI caCI2 MgCI, LaCI3
2
-7
4
ANS
b
6
2
( M)
-6 -5 - 4 -3 - 2 -1
plicalbe to this binding. Since the fluorescence intensity is proportional to the amount of bound ANS, we obtain KC I = qN(1) 1+KC where q is a proportionality constant related to quantum yield, N , the maximum number of the sites, and C, the concentration of free ANS. Equation 1 is recasted as
0
l o g [salt3 ( M )
Figure 1. Fluorescence intensity of ANS added to liposomal suspension as a function of molar concentration of various salts. The total concentratlon of ANS is flxed at lo4 M. Figure l a shows the data of PC and lb, those of PE: (0)NaCI; (0),KCI; (O), CaCI,; ( 0 )MgCI,; (0) Lac&.
counted. The chemotactic response, R, was represented as R = (nl - nz)/(nl + nz) All measurements were performed at 22 "C.
Results and Discussion Characterization of ANS Fluoresence by Use of Liposomes.6 Figure 1 shows fluoresence intensity, I , of ANS added to PC- and PE-liposomes suspended in various salt solutions. The ANS was added into the liposomal suspension at a constant concentration of M. The effectiveness of salt species on enhancement of the fluorescence intensity is in the order Lac& > MgC12 CaC12 > NaCl = KC1. Since the quantum yield of ANS is very small in hydrophilic environments, the fluorescence of ANS in the liposomal system is attributed mainly to ANS molecules bound to the liposome surface. In fact, the fluorescence intensity was proportional to the amount of ANS bound. The proportionality constant depended only on the ionic strength and was independent of the ion species involved. The amount of ANS bound to the liposome increases with increasing salt concentration. The presence of multivalent cations also facilitates binding of ANS to the liposome. Figure 2 represents the relation between fluorescence intensity, I , and ANS concentration added to PC- and PE-liposomes in 100 mM NaC1. At high ANS concentrations, the fluorescence intensity approaches a plateau, which suggests the existence of the maximum quantity of bound ANS. The Langmuir adsorption isotherm is ap(6) Kamo,N.; Aiuchi, T.;Kurihara, K.; Kobatake, Y. Colloid Polym. Sci. 1978,256, 31.
showing that values of qN and K can be determined from a double reciprocal plot between I and C. In eq 1, K is given by K = exp(-AG/RT) (3) where AG is the change in the electrochemical free energy associated with the binding, and R and T have their usual thermodynamic significance. Since ANS bears a negative electric charge, AG is composed of two terms as follows: AG = AGO- F$ (4) where AGO represents the nonelectrical part of the freeenergy change due to the binding, and $, the electrostatic potential difference between the binding site and the bulk solution. X-ray analyses7 suggest that ANS sticks in the polar head group of the membrane. Hence it is reasonable that $ is the potential difference between just inside of the membrane and bulk solution, Le., the surface potential. It is, however, not measurable experimentally for liposomes or living cells. Thus, the zeta-potential of the liposomes, f, is used as an experimental approximation to $; thus AG = AGO- Ff (5) K = K'exp(Ff/RT) K ' = exp(-AGo/RT) (6) Figure 3, a and b, shows the zeta potentials of liposomes suspended in a variety of salts, indicating that multivalent cations decrease the zeta potential (deflects to positive direction) at lower concentrations. Figure 3c is a double reciprocal plot between I and C for the same system as in Figure 3, a and b, where two different concentrations of ANS were used. Note that these data fall on a single straight line for respective PC- and PE-liposomes. The variation of the abscissa is attributed to the change in the surface or zeta potential due to the change in salt concentration. When the fluoresence intensity is measured under varying concentrations of ANS (C in eq l),the value of K' (7) Lesslauer, W.; Cain, J.; Blasic, J. K. Biochim. Biophys. Acta 1971, 241, 547.
The Journal of Physical Chemistty, Vol. 85, No. 22, 198 1 3297
Chemoreception in Tetrahymena pyriformis a
> E
TABLE I : Comparison of Changes in the Surface Potential ( A , mV) Determined by Two Methods'" f potential, A , 10+K, A , chemical stimuli mV mV M-' mV - 64.6 1.47 +10 mM NaCl -51.7 12.9 2.56 14.1 8.6 2.09 -56.0 9.0 +100pM CaC1,
#
0
-.-m
Y
..
E -20 0)
a The f potential was determined by electrophoresis. K is the ANS binding constant to the liposome, Changes in the surface potential were estimated according to eq 6 (see text).
0
8f -40 -60 -6 -5 -4
-3 - 2 - 1
log [salt]
0
(M)
5
E
PE
I
NH&I NaCl
- 0.5
b
0-
KCI
'
I
-0
-7
-1.01
-6 logC
Y
.-m
c)
E
-5
-4 ( M I
LiCl
-2
-3
Flgure 4. Chemotactic response, R , as a function of the concentration of inorganic salts.
-20-
0)
m ,.I
'-
j
-40-
t,+x I
0 LaCI3
,
,
,
-60 -6
0 CaC$
MgC12
0 KCI
o NaCl OKCI
40
-5 -4 -3 - 2 - 1 0 log [ s a l t ] ( M )
1.6
1.2
-7
-6
-5 log
c
-4 -3 ( M I
-2
Flgure 5. Changes in the fluorescence intensity ( A f )of an ANS-T. pyriformis suspension as a function of the concentration of inorganic salts. .4
2
4
6
8
10
( x ) e x p ( -F5 e) x
Flgure 3. Zeta potential of liposomes as a function of molar concentration of various salts: (a) PC; (b) PE. Notations are the same as those in Figure 1. (c) Plots of 111 against ( 1 I C ) exp(-F{/RT) according to eq 2.
exp(Fr/RT) can be determined from the double reciprocal plots. With values of K' exp(F{/RT) obtained a t two different conditions, e.g., different salt concentrations, the changes of surface or zeta potential can be estimated provided that nonelectrical parts of free-energy changes in ANS binding are equal.8 In Table I, the changes in zeta potential estimated by the ANS fluorescence method described above are compared with that measured directly by efectrophoretic mobility. The agreement is fairly good, indicating that we can estimate the zeta or surface potential change of the sample which is inaccessible to direct electrokinetic measurements. In addition, even in the presence of isoamyl ~
(8) Tanabe, H.; Kobatake Y. Submitted to Biochemistry.
acetate, which is supposed to bring about membrane conformational change (see later), the agreement is good. In a previous paper? we showed that eq 1 is applicable to the analysis of ANS fluoresence change in mitochondria in association with energization of the membrane. Application of ANS Fluorescence to T. pyriformk8 In the previous section, it was shown that changes in ANS fluorescence reflect those in the surface potential. Next, we will apply the ANS fluorescence to the study of chemoreception in T. pyriformis. Figure 4 represents the chemotactic responses of T. pyriformis to inorganic salts. The cells exhibit negative chemotaxis to all the salts examined above the respective threshold concentrations (Chi& The magnitude of the response increases linearly with logarithmic concentration of chemical stimuli. The thresholds of salts of multivalent cations are lowered with an increase of valence of cations. No significant difference is found between CaClz and that of MgC1,. Note that the results in Figure 4 are quite similar to the zeta potential of liposomes shown in Figure 3, a and b, except for monovalent cations. The threshold concentrations of monovalent cations vary in the order NH&l N NaCl> LiCl > KC1. (9) Aiuchi, T.; Karno, N.; Kurihara, K.; Kobatake, Y. Biochemistry 1977, 16, 1626.
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The Journal of Physical Chem/stty, Vol. 85, No. 22, 1981
TABLE 11: Fluorimetric Estimation of the Change in the Surface Potential in 2'. pyriformis in Association with Chemoreception
I -4 -31
chemical stimuli
h
v
i10
mM NaCl isoamyl acetate
i5 mV
10-4K,M-' 3.32 4.00 4.28
A , mV
4.8 6.5
0.24 -
-6
-7
log
-4 - 3
-5
.-0 m
0.20-
-m
-
I
Figure 8. Plots of CFANs against C,,,, for respectlve inorganic salts.
40
-
C
CFS( M )
.-
L
0
t
4-
n
menthol
4
isoamyl acetate
0.16
t
isoamyl acetate
'6 I \
5
1 2 Concn.
5 10 20 (mM)
50
Figure 8. Dependence of the fluorescence polarization on the concentration of isoamyl acetate and 1-butanol. The bars represent the range of experimental variation.
-5 log
-4
-3
C
( M
-2
-1
1
Figure 7. Changes in the fluorescence intensity (Af) of an ANS-T. pyrifomis suspenslon as a function of the concentrationof hydrophobic compounds which are so-called "odorants" for higher vertebrates.
Figure 5 represents the change in the fluorescence intensity of an ANS-cell suspension as a function of the concentration of salts. The fluorescence intensity increases with increase of salt concentration. The concentration where the fluorescence intensity starts to change (CFANS) was lowered with increase in the valence of the salts. No difference was found between salts of the same valence such as NaCl and KC1 or as CaClz and MgC12. In Figure 6, CFANsis plotted against Ctaxisfor respective salts. A linear relationship is found between both values except for KC1. T. pyriformis exhibits negative chemotaxis to various hydrophobic compounds, which are so-called odorants for higher vertebrates. Figure 7 shows the ANS fluorescence vs. concentration of such chemicals. As with inorganic stimuli, the fluorescence intensity starts to change above certain threshold concentrations of chemicals, and increases with increase in their concentrations. The values of CFANsfor these compounds are approximately equal to &is, too. Since T. pyriformis swims and the organism exhibits galvanotaxis (taxis by external electric field), we cannot perform the electrophoretic experiments to determine the surface potential change. According to eq 1, the ANS fluorescence may be changed by the change of either qN or K. The value of qN can be evaluated by extrapolation to the ordinate intercept of 1/1vs. 1/C plots. The values of qN are equal for cases with and without chemical stimuli, suggesting that the change in ANS fluorescence is attributed to the change of K. Equation 6 shows that K is composed of nonelectrical and electrical parts of free-energy change. In order to separate these two terms, we employed N-phenylnaphthylamine (NPN) which is a fluorescent analogue of ANS that lacks the sulfonyl group. Thus, the free energy change in NPN binding stems only
from the nonelectrical term, AGO, in eq 4. No NPNfluoresence change was observed when stimulus chemicals were added to the medium. These facts imply that the ANS fluorescence associated with the chemoreception in T. pyriformis can be attributed to the change of the surface potential. As described in the previous section, from the slope of 1 / Z vs. 1/C plot, we can estimate the change of the surface potential. The results are shown in Table 11. Structural Change in Association with Chemoreception.'O The good agreement between CpANS and Chis suggests that the change of the surface potential is brought about by the reception of chemicals. The surface potential is a function of ionic strength and surface charge density. Since electrically neutral compounds do not change the ionic strength of the medium, it is likely that the decrease of the surface potential that accompanies reception of hydrophobic compounds (odorants) is caused by a decrease of the surface charge density. Probably, conformational changes of the receptor membrane caused by the adsorption of the hydrophobic compounds lowers the surface charge by burying the negative charge of the membrane surface into the interior of the membrane or by exposure of the positive charge on the membrane surface. To proceed further, we measured the change in fluidity of the membrane in response to chemical stimuli by use of DPH. In Figure 8, the P value is plotted against the concentration of isoamyl acetate and 1-butanol. The P value in the absence of chemical stimuli was 0.242 f 0.010. This is close to the value determined for the isolated membrane fraction of the cell and suggeststhat the P value in the present experiment is that of the surface membrane irrespective of use of whole cells. The fluorescence polarization decreases linearly with an increase of added chemicals. It phould be noted that the concentrations of the chemicals at which the fluorescencepolarization starts to decrease are practically identical with respectvie Ctaxb As shown in Figure 8, the P value is a function of concentration of chemical stimuli. Table I11 compares the P (10)Tanabe, H.; Kurihara, K.; Kobatake, Y. Biochemistry 1980,19, 5339.
The Journal of Physical Chemistry, Vol. 85, No. 22, 1981 3299
Chemoreception in Tetrahymena pyrlformis
'
TABLE 111: Fluorescence Polarization of DPH Measured in the Presence of Hydrophobic Compoundslo chemical
concn: M
control 1-propanol 1-butanol isoamyl acetate l-menthol 1-heptanoic acid p-ionone
2 x 10-l 3x 2x 5x 2X 5x
polarizationb 0.242 f 0.210 f 0.205 f 0.195 ?: 0.166 * 0.198 * 0.220 f
0.010 0.001 0.005 0.005 0.001 0.005 0.005
a The concentrations are 10 times the chemotactic threshold concentrations for respective chemicals except for p-ionone. In the case of p-ionone, the concentration is 250 times the chemotactic threshold. Each value is the average of two or three determinations. The value of the control is the average of all determinations.
values in the presence of chemical stimuli of 10 times the chemotactic threshold concentrations. In the case of 0ionone, the P value at 250 times threshold is listed since &ionone gives only a small change in the value. Application of all the hydrophobic compounds examined decreased the fluorescence polarization, but the degree of change varies among the chemicals. On the other hand, inorganic salts such as NaC1, KC1, and CaC12 did not change the fluorescence polarization, although these inorganic salts induced negative chemotaxis in T. pyriformis. Inorganic salts seem to interact directly with the membrane surface and induce the change in the surface potential. Membrane Potential Change in Association with Chemoreception.ll Rh6G is a membrane-potential sensitive probe. When the membrane potential is set up across the liposomal membrane with a K+ ionophore, valinomycin, and K+, the fluorescence of Rh6G corresponds to the membrane potential calculated with the Nernst equation for K+. Rh6G fluoresces in a hydrophilic medium, and its fluoresence is quenched in hydrophobic moiety of membranes. When cells are hyperpolarized, Rh6G (having a positive charge) is accumulated into the cells because it is considered to be permeable to the membrane. The accumulation may result in an increase of Rh6G concentration in the membrane phase which decreases the fluorescence intensity. As the concentration of stimuli increased beyond a threshold, Rh6G fluorescence started to increase (implying the depolarization of the cell). This threshold concen(11)Aiuchi, T.; Tanabe, H.; Kurihara, K.; Kobatake, Y. Biochim. Biophys. Acta 1980,628,355.
trations agreed approximately with Cw It is noted that this coincidence was obtained in the case of KC1, where disagreement was observed in ANS fluoresence (surface potential probe). This finding suggests that ANS is not so sensitive a probe for the membrane potential as has been stressed by some authors. Valinomycin has at most a slight effect on Rh6G fluorescence, implying that the T. pyriformis membrane at rest is permeable to K+. This may be the main cause of the discrepancy between CFANsand Ctaxis.
Concluding Remarks T. pyriformis exhibits chemotaxis to various stimuli by regulating its ciliary motion. The negative chemotactic responses to various chemical compounds were accomplished by modulating the frequency of alteration in swimming direction. The initial step of the chemoreception is the adsorption of chemical stimuli on the receptor membrane. The membrane lipids may be one of the candidates of receptors for inorganic salts and hydrophobic compounds (odorants). In fact, we have shown that the change in the lipid composition of T. pyriformis affects the sensitivity of the chemotactic responses against the above stimuli. But, lipids are not the only receptor molcules, nor are they enough to induce the sensation as is evident from the fact that isoamyl acetate cannot change the ANS fluorescence of liposomes made of lipids, while this chemical can change the fluorescence of ANS-cell suspension. The adsorption of hydrophobic compounds on the membrane alters the fluidity of the lipids, which induces the conformational change of proteins or lipids in the membrane and changes the surface potential. The significance of the surface potential in chemoreceptive membranes has been stressed in our previous papers.12J3 Several other investigators pointed out the importance of surface potential to the membrane potential of living and model membranes.14J5 In Paramecium, depolarization of the cell results in the influx of Ca2+,which regulates the ciliary motion.16J7 A similar mechanism is considered to occur in T. pyriformis. In fact, we showed that the presence of Ca2+in the external medium is absolutely necessary for chemotaxis in T. pyriformis. (12)Kamo, N.; Miyake, M.; Kurihara, K.; Kobatake, Y. Biochim. Biophys. Acta 1974,367, 1, 11. (13) Kurihara, K.; Kamo, N.;Kobatake, Y. Adu. Biophys. 1978,10,27. (14)Chandler, W.K.; Hodgkin, A. L.; Meves, H. J. Physiol. 1965,180, 821. (15) McLaughlin, S.; Harary, H. Biophys. J. 1974,14,200. (16)Eckert, R.Science 1972,176,473. (17)Eckart, R.;Naitoh, Y.J. Protozool. 1972,19,237.
