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Photoisomerization of Retinal at 13-Ene is Important for Phototaxis of Chlumydomonas reinhnrdlii: Simultaneous Measurements of Phototactic and Photophobic Responses Tetsuo Takahashi*, Kazuo Yoshihara*, MasakatsuWatanabe#,Mamoru Kubotag, Randy Johnson”, Fadila Derguini” and Koji Nakanishi”* * Suntory Institute for Bioorganic Research,Shimamoto-cho,Mishima-gun, Osaka, 618 Japan # National Institute for Basic Biology, Okazaki National ResearchInstitutes, Okazaki, 444 Japan * Department of Chemistry, Columbia University, New York, NY 10027

Received

July

5,

1991

Summary:

A real-time automatedmethodwas developedfor simultaneousmeasurements of phototactic orientation (phototaxis) and step-up photophobic response of flagellated microorganisms. Addition of all-rrans retinal restoredboth photoresponsesin a carotenoiddeficient mutant strain of Chlamydomonasreinhardtii in a dose-dependentmanner. The phototactic orientation was biphasic with respect to both the light intensity and the concentration of retinal. All-vans retinal wasmore effective than 11-cisretinal to regenerate both photobehavioral responses.Analogs having locked 1I-cis configurations and a phenyl ring in the sidechain also inducedphotoresponses,although at concentrationsmore than two ordersof magnitudehigher than all-truns retinal. According to the presentassaymethod, the responseswere hardly detectablein cells incubatedwith retinal analogsin which the 13-ene was locked in either its trans or cis configuration. The results strongly suggestthat the isomerization of the 13-14 double bond is important for photobehavioral signal transduction and that a single retinal-dependentphotoreceptor controls both phototactic and photophobic responses. 0 1991 Academic Press, Inc.

Using a carotenoid-deficient blind mutant FN68 of the motile greenalga Chkzmydomonas reinhardtii, Foster et al. showedthat retinal is the chromophoreof a sensoryphotoreceptor;in thesestudiesthe rate of migration of a cell population away from an actinic light sourcewas measured(1). It was alsoshown that a variety of retinal analogswere incorporated into the cell, functioned as its photosensory chromophore, and induced negative phototaxis. The analogswhich inducedphototaxis included thosein which cisltrans isomerizationsat 9-, 1l-, or 13-eneswere blocked, respectively, by extra phenyl, 7- or 5-memberedrings, and it was concludedphototaxis involved no isomerizationof specific double bonds(2,3). However, since no other rhodopsin functions without photoisomerization, the earlier experimentshave beenrecheckedwith a different carotenoid-deficientmutant strain CC-2359 and a newly designedcomputerizedmovement-trackingsystemwhich enablesone to measure both the phototactic (orientation) and photophobic (“stop”) responsessimultaneously.

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0006-291x/91 $1.50 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

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Current results basically agree with earlier results, including the phototactic responses exhibited by the 1 l-cis -locked isomers; however, the present results strongly suggest that alltruns retinal is the natural chromophore and that isomerization of the 13-ene is crucial for the functioning. MATERIALS

AND

METHODS

Chlamy&monas reinhardtii strain CC-2359 @s-l, a carotenoid-deficient strain, originally selected by Iroshnikova et al., 4) was from John Spudich. The strain was grown and resuspended in the dark in a standard medium (5,6), the cells were diluted with the medium and used throughout the experiment without further treatment. All-pans retinal (Sigma Chemical Co.) and retinal analogs (7,8,9) were purified prior to usage by HPLC Cosmosil 10 x 300 mm (Si-60, Nacalai Tesque, Inc., Kyoto), eluted with 10 % ethyl ether in hexane, flow rate 6 ml/rnin, and added to the cell suspension as an ethanolic solution. The final concentration of the solvent was always kept at ~0.1 % (v/v). Simultaneous measurements of phototaxis and photophobic reaction. A quartz chamber (10 x 10 x 0.5 mm) filled with 100 ~1 of a cell suspension (ca. 1Oacells/ml) was sealed with a coverslip and set under a microscope (Olympus, Tokyo) equipped with a 4 x objective and a 3.3 x relay lense which projects real images of the specimen onto a CCD video camera (XC-77 or XC-77CE, Sony Corp, Tokyo). To obtain uniform dark-field illumination over a wide field, a Ph-3 phase-contrast condenser was used for infrared ( k > 850 nm) observing light. Temperature was kept at 21-22 “C. A video-digitizer board (Keio Electronic Industry Co., Ibaraki, Osaka) along with a HD64180 CPU board (EM3-Star, Megasoft Co., Osaka) was linked to a computer (PC9801, NEC, Tokyo) so as to digitize and process video-images. The system digitizes and transfers 5 frames/second to the host computer, as binarized data of 256 x 256 pixels. Principle of the fast detection of the displacement of a moving object between two consecutive frames is described elsewhere (10, 11). Timing for actinic irradiation with monochromatic light (bandwidth 1 nm) from Okazaki Large Spectrograph (OLS in National Institute for Basic Biology; 12) was controlled by an electronic shutter (Copal Co. Ltd., Tokyo) interfaced to the computer. Usually 3 equivalent systems were aligned at desired positions (wavelengths) of the OLS. Light intensities (fluence-rates) were changed with neutral density filters, and measured with a calibrated silicon-photodiode radiometer. Timing for exposure to the actinic light and the data acquisition for measurement of both phototactic and photophobic responses are illustrated in Fig. 1A. For each specimen, the onphobic

response

Fig. 1. A. Timing chart for actinic irradiation and data acquisition.Trajectoriesof

microorganisms were recordedin six consecutivedigitized framesat framerate of 5 s-1 during the periodsindicatedby W. A computersoftwareautomaticallydetectsthe displacement of eachindividualcell track betweentwo consecutive framesandthedatawere accumulated.B: Histogramsof the two-dimensionaldisplacementper 200 ms of the Chlamyabmonas cellsincubatedwith (upper)or withoutexogenous retinal(lower). ,&rrows indicate the direction of the actinic light. C: The histogramwas then divided into 5 subdomains.Phototaxisindex andthepercentof the cellsshowingphotophobicresponse werecalculatedasdescribed in the text. 1274

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off cycle was repeated 9 - 14 times during the 15 min period of the measurement, during which time 1500 - 2000 counts of two-dimensional displacements of the cells per 200 ms were obtained. The period of actinic irradiation was 17 s except for experiments shown in Fig. 3 and Table 1. The resultant velocity table (Fig. 1B) was divided into 5 clover-leaf subdomains (Fig. lC), and a phototaxis index was calculated as the integration of the counts over the domain 1 divided by that over domains 1, 2, 3, and 4. The magnitude of photophobic response was calculated from the number of cell tracks whose displacement falls in subdomain 0 after actinic irradiation. Percent phobic response (P) was normalized as P = 100 x (F-S)/( l-S), where F and S represent, respectively, the fraction of stopped cells after a stimulus and that of cells showing spontaneous stop without the stimulus. RESULTS

AND

DISCUSSION

In the present paper, we adopt the definition for the terms phototaxis and photophobic response as spelled out by Diehn et al. (13): (i) Phototaxis is a movement oriented with respect to the stimulating light direction; therefore in phototaxis, it is necessary for the microorganism to detect the light beam axis. (ii) Photophobic response, in contrast, is a reaction to a temporal change in actinic light intensity, and normally consists of a brief stop, followed by a random change in the swimming direction. The phototactic and photophobic responses, both have been measured separately and simultaneously in this study, were hardly detectable when the mutant strain CC-2359 was incubated without

exogenous retinal and subjected to 400 - 530 nm actinic light with an

intensity less than 7 x 1013 photons/mm2s.

However,

the cell restored both phototactic and

photophobic responses soon after addition of all-rrans retinal. These responses were plotted against intensity of actinic light at three wavelengths (Fig. 2A). The phototaxis index, which represents the biased orientation of the swimming

direction in a cell population, exhibited

biphasic curves; namely, the cells showed positive phototaxis (swim toward the light source) at low light intensity, whereas at high actinic intensity they showed negative taxis (swim away from the light source). Moreover, in cases of an abrupt irradiation, the cell exhibited phobic responses at intensities slightly higher than those where the phototaxis indexes peaked. A photoresponse

is, in general, a function of both the light intensity and the receptor

concentration; namely, .. 1 R = f($e.I.N), where letters represent the followings: R, the magnitude of the photoresponse; $, the quantum efficiency photoreceptor

in photosignal

transduction;

o, the capture cross section of the

pigment; I, light intensity; N, number of receptor molecules in a cell (14).

Since N is variable according to the concentration of the exogenous chromophore in the present experiments, the biphasic character is also expected in retinal dose-response curves under unsaturating conditions. To test whether the phototactic and phobic responses are mediated by the same photoreceptor, dose-response curves were measured after different periods of incubation with all-truns retinal I, namely, after 24 hrs incubation (Pig. 2B) and e 15 min incubation (Fig. 2C). Independent of the incubation period, the photophobic response appeared as, and only when, the phototaxis index stopped increasing; moreover, the maximal value of the ~phototaxis index is independent of wavelength, retinal concentration, or reconstitution period. These observations reti&&pena!entphotoreceptor controls bothphotobehmtiors. 1275

strongly suggest that a single

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j:I::-fii tWz 450 nm

L m 0.4

I450

60

nm

I

.

.

22 0.2 2 55 0 E 1o’oo 10” 1 s

AND BIOPHYSICAL

40

20

0

ld*

495nm

10’3

10’4

r

0

nm

60

Ok

0.2 t

Intensity

(photons

/mm%)

log [all-trans

retinal

I

log [ all-trans

retlnal

1

Fin. 2. Dependence of phototaxis index (closed symbols) and photophobic response (open symbols) on actinic irradiance (A) or on concentration of exogenous all-truns retinal (B, C). A: All-tranr retinal (6.2 x IO-* M) in ethanolic solution was added to a diluted cell suspension to give a final concentration of 4.7 x lO-*l M and incubated for 6 hrs at 21 “C before each measurement. B: Cells were incubated with all-tram retinal (circles) for 24 hrs before each measurement. C: Cells were incubated with all-rruns retinal for 10 min (405 nm), 15 min (450 nm), or 20 mm (495 nm) before each 15 mm measurement (circles). Independently, cells were incubated with 5 x lO-3 a.u. of either analog 2 (squares) or 3 (triangles) for 6 hrs, all-rruns retinal was then added to the medium, and the responses were measured 10 min (405 nm), 15 min (450 nm) or 20 min (495 nm) later. Broken arrows indicate the inhibitory effect of analogs 2 or 3 on photophobic response. Actinic intensities for panels B and C: 7.0 x 1013, 1.0 x 1Ol4 and 1.1 x lOI photons/mm*s for 405 nm, 450 nm and 495 nm, respectively.

Yan et al. reported inhibition of photophobic response of the archaebacterium Halobacterium halobium by retinal analogsin which cisltrans isomerization at 13-14double bond was blocked (15). Lawson et al. have shown that these analogs also inhibit photophobic response of Chlamydomonas (Lawson, M., Zachs, D. N., Derguini, F., Nakanishi, K., Spudich, J. L., submitted); in contrast, earlier studies (2, 3) showed that migration of Chlamydomonas cells away from the light source does not require specific double bond isomerization, including the 13, 1Cene. As described below, the present simultaneousmeasurementsof both phototactic and phobic responsesprovide a clue to an understandingof this discrepancy. Neither significant phototactic orientation (index > 0.29) nor photophobic responseswere encountered when the cells were incubated with analogs 2 and 3 even at the highest -3 concentration (5 x 10 absorbanceunit) when exposed to an actinic intensity of lessthan 1014 photons/mm2s. All-trans retinal was then added,and the behavioral responseswere measured10 - 20 min later (Fig. 2C). As indicated by black circles (all-trans retinal alone), squares(analog 2 ) and triangles (analog 3 )(Fig. 2C), the phototaxis indexes were not affected significantly by preincubation with locked analogs 2 and 3. However, the photophobic responseis greatly reducedby preincubation with 2 or 3 asindicated by broken arrows.

The generaltrendsof changesin the phototaxis index and the phobic responseare in 1276

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(I)

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(2)

1 3-&locked

(3 )

good agreement and show that the effective concentration of all-trans retinal was an order of magnitude reduced by the locked analogs. This leads to the conclusion that the locked analogs are indeed incorporated

into the photoreceptor

and that their efficiency

in mediating the

photoresponse is negligibly small, thereby suggesting that cisltrans isomerization at 13-ene is important

(Scheme 1).

We have also incorporated

1 1-cis locked analog 4 ,9, 1l- dicis locked analog 5 and the

phenyl analog 6 into the cells (Table 1). Interestingly, all analogs induced phototactic responses, although the critical concentration of analogs 4 and 5 for inducing phototaxis or photophobic response is much higher (1000 - 10000 fold) than that of all-tram retinal. Note that the earlier studies (2, 3) employed even higher analog concentrations (ca. 80 fold as compared to the highest concentration in the present experiments).

It should also be noted that

one must discriminate the efficiency in transduction from the critical chromophore concentration for the responses; the former corresponds to +a in Eq. 1, while the latter presumably reflects the affinity to the apoprotein. Of the three analogs shown in Table 1,

Table

1.

Phototactic

and photophobic phototaxis

11 -cis-ret-7

(4)

responses index#

of cells

incubated

/ photophobic

with

analogs

4 - 6*

responses

9,11-dicis-ret-7

(5)

9-12-phenyl-ret

(6)

3nM

0.249 + 0.07 / < 1 %

0.325 f 0.05 /

< 1%

0.318 + 0.003 /

< 1%

30 “M

0.303 * 0.012 / < 1 %

0.301 f 0.05 I 14 f 4 %

0.328 f 0.015 /

< 1%

0.426 It 0.002 / 4 f 3 %

0.245 f 0.01 I43

0.324 f 0.003 I 2 + 2 96

300 “M * *

f 6 4%

Cells were reconstituted with analogs for 3 hrs. Analogs 4 and 5 can undmgo cislmns isomerizatioa around the 13-enc as depicted by the dashed fines, whereas this isomerization is hi&ted in the phenyl analog 6 (9). # Values obtained after 5 s periods of a&tic irradiation (495 nm, 1.7 x 1013 photons/m&) are shown wiih f S.E. (2 or 3 experiments). B Percentage of cells that showed phobic response to tbe onset of the actinic light. l Under the same conditions, 0.03 IM of all-nanc retinal I ,0.24 f .02 / 56 f 2 %.

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analog 5 has the lowest critical concentration (30 nM) for inducing the photophobic response. The phototaxis index of analog 5 apparently shows the tendency toward negative taxis and gives a phototactic response curve similar to that seen in Fig. 2C (495 nm). On the other hand, the phototaxis index of analog 4 still increases at 300 nM, where the phobic response is not yet apparent. These differences in behavioral responses between 4 and 5 probably arise from tbe large difference in the conformations of tbe nonplanar 7-membered rings. The low values of the phototaxis index and the insignificant photophobic response in the phenyl analog 6 may be attributable to a steric hindrance between 11-H and 15-H in its 13-cis configuration (9). Therefore, the results also favor the conclusion that the isomerization at 13-ene is important for significant responses; however the fact that these analogs elicit phototaxis is in basic agreement with earlier reports (2,3). Recent studies of retinal extraction from Chlumydomonas cells have shown that the cells predominantly

contain all-truns

retinal (16). In addition, a spectroscopic

incorporation experiments with 6-s truns and 6-s cis locked retinals (Lawson,

study (17) and M., Zachs, D.

N., Derguini, F., Nakanishi, K., Spudich, J. L., submitted) also suggested an all-truns and a coplanar conformation for the chromophore of Chlumydomonus rhodopsin. To test whether the functional chromophore is all-truns or 11-cis as suggested by earlier studies (2, 3), we have incubated the cells with either all-truns or 1 1-cis retinal at various concentrations and measuredthe photoresponses (Fig. 3). All-rruns retinal was almosttwo ordersof magnitude more effective than 11-cis retinal, as judged from the critical concentration for the photoresponses,strongly suggestingthat the configuration of the functional chromophoreis all-trans. At present,it is unclearwhetherthe remainingactivity of 1l-cis retinal wasclueto a trace amountof the all-rrunsisomerproducedduring the experimentalperiods. The results presented in this paper strongly suggestan all-truns configuration of the chromophoreretinal and that its isomerizutionto I3-cis is critically important for biological function of this eucaxyotic rhodopsin. Further detailed studies are in progress on the photobehavioralresponsesinducedby analogsincluding 11-cis and l l-trurrs locked retinals.

logketinall Fig. 3. Dependence of photoresponses on concentrationof all-rrans (squares)or 11-cis retinal (circles). Cellswereincubatedfor 25 min with retinalsbeforeeachmeasurement. Phototaxisindexrepresents theorientationof theswimmingdirectionof cellsafter5 s period of irradiationwith 495nm actiniclight (1.7 x 1013photons/mmas). 1278

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ACKNOWLEDGMENTS: We thank John Spudich and Moira Lawson for the strain CC2359 and Noriko Sekiya for reading the manuscript. Inhibition experimentson photophobic responseby 13-ene-lockedanalogswere initiated by M.L. and J.S. (Albert Einstein College of Medicine, NY). The study has been partially supportedby NM grant GM 36564 and NIBB cooperative researchprogramfor the OLS (90-520 and91-5 11).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Foster, K. W., Saranak, J., Patel, N., Zarrilli, G., Okabe, M., Kline, T., and Nakanishi, K. (1984) Nature 311, 756-759. Foster, K. W., Saranak, J., Derguini, F., Rao, V. J., Zarrilli, G. R., Okabe, M., Fang, J-. M., Shimizu, N., and Nakanishi, K. (1988) J. Am. Chem. Sot. 110, 65896591. Foster, K. W., Saranak, J., Derguini, F., Zarrilli, G. R., Johnson, R., Okabe, M., and Nakanishi, K. (1989) Biochemistry 28,819-824. Iroshnikova, G. A., Rakhimberdieva, M. G., and Karapetyan, N. V. (1982) Soviet Genetics 18, 1350-1356. Sueoka, N., Chiang, K. S., and Kates, J. R. (1967) J. Mol. Biol. 25, 47-66. Hunter, S. H., Provosoli, L., Schatz, A., and Haskins, C. P. (1950) Proc. Am. Phil. Sot. 94, 152-170. Akita, H., Tanis, S. P., Adams, M., Balogh-Nair, V., and Nakanishi, K. (1980) J. Am. Chem. Sot. 102,6370-6372. Fang, J. -M., Carrikar, J. D., Balogh-Nair, V., and Nakanishi, K. (1983) J. Am. Chem. Sot. 105,5162-5164. Kolling, E., Gartner, W., Oesterhelt, D., and Ernst, L. (1984) Angew. Chem. Int. Ed. Engl. 23, 81-82. Takahashi,T., and Kobatake, Y. (1982) Cell Struct. Funct. 7, 183-191. Takahashi, T. (1991) in Image Analysis in Biology (D. -P. Hader, Ed.), CRC Press, Boca Raton, in press. Watanabe, M., Furuya, M., Miyoshi, Y., Inoue, Y., Iwasaki, I., and Matsumoto, K. (1982) Photochem.Photobiol., 36, 491-498. Diehn, B., Feinleib, M., Haupt, W., Hidebrand, E., Lenci, F., and Nultsch, W. (1977) Photochem.Photobiol. 26,559-560. Hartmann, K., M. (1983) in Biophysics (W. Hoppe, W. Lohmann, H. Markl, H. Ziegler, Eds.), pp. 115-144, Springer-Verlag, Berlin. Yan, B., Takahashi, T., Johnson, R., Derguini, F., Nakanishi, K., and Spudich, J. L. (1990) Biophys. J. 57, 807-814. Derguini, F., Mazur, P., Nakanishi, K., Starace, D. M., Saranak, J., and Foster, K. W. (1991) Photochem. Photobiol., in press. Beckman, M., and Hegemann,P. (1991) Biochemistry 30,3692-3697.

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Photoisomerization of retinal at 13-ene is important for phototaxis of Chlamydomonas reinhardtii: simultaneous measurements of phototactic and photophobic responses.

A real-time automated method was developed for simultaneous measurements of phototactic orientation (phototaxis) and step-up photophobic response of f...
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