j cell biol 1985 johnson 1435 46

8/8/2019 J Cell Biol 1985 Johnson 1435 46

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Compartmentalization of Algal Bioluminescence:Autofluorescence of Bioluminescent Particles in theDinoflagellate Gonyaulaxas Studied with Image-intensifiedVideo Microscopy and Flow Cytometry

CARL HIRSCHIE JOH NSON ,* SHIN YA INOUI~, II ALA N FLINT, and J. W. HASTINGS**Biological Laboratories, Harvard Un iversity, Cambridge, Massachusetts 02138; UMarine BiologicalLaboratories, Woods Ho le, Massachusetts 02543; and SChildren "s Hospital, Harvard Medical School,Boston, Massachusetts 02115

ABSTRACT Comp artmen talization of specialized function s to discrete locales is a fundamentaltheme of eucaryotic organization in cells. We report here that bioluminescence of thedinoflagellate alga Gonyaulax originates in vivo from discrete subcellular loci that are intrinsi-cally fluorescent. We demonstrate this localization by comparing the loci of fluorescence andbioluminescence as visualized by image-intensified video microscopy. These fluorescentparticles appeared to be the sam e as the previous ly described in vitro "scintillons." Weattribute the endogenous fluorescence to that of the bioluminescence substrate, luciferin,because (a) the fluor esce nce excitati on and emission characteristics are comp arable , (b) theautofluorescence is lost after exhaustive stimulation of bioluminescence, and (c) the fluores-cence of discharged particles in vitro can be restored by adding luciferin. T he fluorescence invivo exhibits a standard pr operty o f circadian (daily) rhythm icity: unde r constant environ mentalconditions, the intensity of the particle fluorescence fluctuates cyclically (it is maximal duringthe nig ht phase and is low during th e day). Thus, luciferin is localized wi thin the cell at discreteloci from which the bioluminescence emanates; the cellular quantity of luciferin is rhythmicallymodulated by the circadian clock.

The abi l ity of "red t ide" a lgae to emi t l ight has long fascinatedbiologis ts (16). Al though muc h i s know n ab out the biochem -istry of this algal luminesc ence, the cell biology of the ph e-nom eno n i s less wel l unders tood. In the dinof lagel la te Nocti-luca miliaris, biolumines cent l ight is emi t ted f rom subcel lular"microsources" in response to a membrane depolar izat ion(9), which may activate a proton flux (25); but the triggeringpathway i s incompletely character ized and the ul t ras t ructuralident i ty of these microsources i s unkno wn. In Pyrocystisfusiformis, the int racel lular s i tes of light product ion have beenvisual ized, but the t ransducing mechanism is even less wel lunders tood than in Noctiluca (34, 38, 39).In a thi rd bioluminescent dinof lagel la te , Gonyaulax poly-edra, f ract ionat ion of cel l ext racts revealed sedimentable bio-luminescent particles which were called "scinti l lons" (6, 19).These scinti l lons were defined as self-contained particulatesystems whose in vi t ro l ight product ion i s speci fically depend-

ent upo n protons : ~ l ight emission in vitro oc curs as a flashkinetically similar to the flash in vivo and is tr iggered by arapid pH change (e.g., pH 8 to 6; see references 7 and 18).Ear ly s tudies by image- intensif ied m icroscopy suggested thatf lashes in vivo emanated f rom discrete subcel lular sourcesthat were not dis t r ibuted uni formly throughout the cytoplasm(28). In vitro studies using image-intensified microscopy sup-ported the idea that these sources were identical to scinti l lons(27).

Bioluminescence in Gonyaulax is of special interest beca usei t and other cel lular sys tems are cont rol led by the ci rcadianclock. Major research ef for t s have character ized the b iochem -J Scintillons were originally hypothesized to be particles con tainin gguanine crystals (6, 19). Later studies (12) excluded this proposal anddefined scintillons as sedimentable subcellular particles that are ca-pable of bioluminescent light emission in vitro.

THE JOURNAL OF CELL BIOLOGY VOLUME 100 MAY 1985 1 43 5- 14 46 1435 The Rockefeller Universi ty Press . 0021- 9525]8 5/05] 1435/' 12 $1.00

Published May 1, 1985


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i c al me a n s b y wh ic h th e c lo c k c o n t r o l s r h y th m ic b e h a v io r s inthis alga (22, 29) .

I n th e wo r k r e p o r te d h e r e , we c o n f i r m th a t Gonyaulaxbio luminescence or ig ina tes in v ivo f rom d isc re te subce l lu la rpar t ic les tha t a re in tr ins ica l ly f luorescent . This f luorescence isa t t r ibu tab le to the b io luminescent subs tra te , luc ife r in , whichi s th e r e f o re c o m p a r tme n ta l i z e d w i th in th e c el l. W e f o u n d th a tluc ife r in conten t o f Gonyaulax ce l ls , measured by the in ten-s i ty of th is f luorescence , is rhy thmica l ly modula ted by thec ircad ian c lock . The f luorescent par t ic les were presen t ine x t r a c ts o f th e c e ll s a n d a p p e a r e d to c o r r e s p o n d to th e p r e v i -ous ly descr ibed in v i t ro sc in t i l lons .M A T ER IA L S A N D M E T H O D SCulture Conditions and Strains: Uni a l ga l , bu t no t axen i c , cu l t u reso f Gon.vaulax were grown a t 20 + 2"C in 2 . 8- l i t e r F e rn bach f l a s ks conta in in g 1l i t e r o f f / 2 me diu m (13) , omi t t i n g the s il ica te a nd ad ding 0 . 5% au toc l avede x t r a ct o f s o il . T h i s m e d i u m c o n s i s ts o f s e a w a t e r s u p p l e m e n t e d w i t h v i t a m i n s ,n i t ra t e , phos pha te , and t race me ta l s . Cul tures were grown on a l i gh t :da rk cyc leof 12 h of li gh t fo l lowed by 12 h of da rknes s ( l i gh t i n t ens i ty = 150 ta e ins t e ins /m2s).

G. polyedra s t r a in s G P 7 0 a n d G P 6 0 ( b o t h l u m i n o u s ) w e re u s e d i n th i s s tu d yas we l l a s two s t ra ins of Gonyaulax tamarensis ( f o r m e r l y G. excavala), w h i c hw e r e o b t a i n e d f r o m D r . D o n a l d A n d e r s o n ( W o o d s H o l e O c e a n o g r a p h i c I n s ti -t u t e , W o o d s H o l e , M A ) : th e l u m i n o u s G T 4 2 9 (Gonyaulax tamarensis var.o:cavala) a n d t h e n o n l u m i n o u s G T P P (Gonyaulax tamarensis var. tamarensis).Fluorescence and Im age-intensi f ied Video M icroscopy:Gonyaulax ce l l s and ex t rac t s were obs e rved w i th 40 /0 . 75 dry or 100/1 . 30 o i limm ers io n objec t ives a t t ached to a Ze i s s IM-35 inv e r t ed ep i f luores cence mic ro-s cope (Car l Ze is s , Inc . , New Yo rk) . The ep i f luores cence exc i t a t i on was gene r -a t e d b y a m e r c u r y l a m p ( H B O 5 0 W ) u s i n g v a r i o u s s e ts o f f l u o r es c e n c e fi lt er s:(a ) EX (ex i t a t i on) 365 /EM (emis s ion ) 420 long pas s (Ze i s s f i lt e r s e t 487702),(b) EX 395 -420 /EM 450 long pas s (Ze i s s f i lt e r se t 487718), and (c ) EX 450 -490 /EM 520 long pas s (Ze i s s f i lt e r s e t 487709). In mo s t cas es, an ad di t i ona ls hor t -pa s s ba r r i e r f i lt e r (Ze i s s fi lt e r K P 560) w as in t e rpos ed to f i lt e r ou t t he redf l u o r es c e n c e f r o m c h l o r o p h y l l . F o r d i r e c t p h o t o g r a p h y o f t h e m i c r o s c o p i ci m a g e , a n O l y m p u s O M - 2 c a m e r a w a s a t t ac h e d t o t h e c a m e r a p o r t o f t h e I M -3 5 m i c r o s c o p e .

I m a g e - i n te n s i f ic a t i o n o f t h e f l u o r e sc e n t a n d b i o l u m i n e s c e n t i m a g e s w a sa c c o m p l i s h e d b y c o u p l i n g a Z e i s s / V e n u s T V - 3 M t h r ee - s ta g e i m a g e - i n te n s i fi e dvid i con cam era to t he c ine -por t on the s ide of t he 1M-35. Af t e r ins e r t i on oft i m e a n d d a t e i n f o r m a t i o n ( M o d e l E T 2 0 2 [ C r a m e r V i d e o , N e w t o n , M A ] ) , th ev ideo s igna l wa s s tored on a 3 /4 inch cas s e t t e w i th a S o ny V O-4800 v ideo t aperecorde r .

T h e t e m p o r a l a n d s p a ti a l p r o p e r t ie s o f Gonyaulax f luores cence and b io lu-m i n e s c e n c e w e r e a n a l y z e d b y t r a n s c r i b i n g t h e r e l ev a n t c a s s e tt e s e q u e n c e o n t oa v ideo mot io n ana lyze r (S ony S VM 1010) . We then ana lyzed f i e ld-by-f i eld theent i re 10-s s equenc e s tored on the d i s k of t he mo t ion ana lyze r . K ine t i c chang eso f t h e b i o l u m i n e s c e n c e o f s in g le s e i n ti l lo n s w e r e m e a s u r e d b y i n s e r ti n g a v i d e oa n a l y z e r (M o d e l 3 2 1 [ C o l o r a d o V i d e o , B o u l d e r, C O ] ) i n t o t h e i n s t r u m e n t a t i o nne two rk tha t a l l owed the in t ens i ty of sing le p ixe l s t o be recorded on a s t r i p -c h a r t r e c o r d er . I n s o m e c a se s, t h e c o m p a r i s o n o f f l u o r e s c e n ce a n d b i o l u m i n e s -cence d i s t r i bu t ions was fac il it a ted by image pro ces s ing us ing a s ys t em func t ion-a l ly i denti cal t o t he IMA GR -I s ys t em f rom In t e rac t ive V ideo S ys t em s (Conc ord ,M A ) . P h o t o g r a p h y o f t h e i m a g e - i n te n s i fi e d i m a g e s w a s d o n e f r o m s i n g le f r a m e so n t h e v i d e o m o n i t o r t h r o u g h a R o n c h i g r a t i n g t o e l i m i n a t e s c a n l i n e s a n di m p r o v e r e s o l u t i o n ( 2 1 ) .Preparation of Cells for Microscopy: F or cor re l a t ing the s ubee l -lu l a r d i s t r i bu t ion s of au tof luores ce nce and s t im ula t ed b io lum ines cenee , ce l lsw e r e i m m o b i l i z e d b y e m b e d d i n g t h e m i n 2 % l o w - t e m p e r a t u r e ge ll in g a g a r o s e( S e a P r e p 1 5 / 4 5 a g a r o s e f r o m F M C C o r p o r a t i o n , M a r i n e C o l l o i d s D i v i s i o n ,R o c k l a n d , M N ) . 1 m l of Gonyaulax ce ll s was added to 1 ml o f 4% aga ros e inf / 2 m e d i u m t h a t h a d b e e n h e a t e d t o 9 0 C t o m e l t t h e a g a r o s e a n d t h e n c o o l e dto 25"C. Af t e r gent l e mix ing of t he Gonyaulax/agarose s u s p e n s i o n , t w o - d r o pa l i q u o t s ( ~ 1 00 u l ) o f t h e s u s p e n s i o n w e r e p l a c e d i n s m a l l w e l l s o n t o p o f 2 4 - x -60 - ra m cover s l i ps (we us ed s ec t ions of 1 . 5 ml p l a s t ic cen t r i fuge tubes to m aket h e w e ll ). T h e p r e p a r a t i o n s w e r e t h e n p l a c e d i n a h u m i d i f ie d c h a m b e r i n t h eda rk a t 18-20"C un t i l t he aga ros e so l id i fi ed . Ag aros e embedd ing was us ua l lyp e r f o r m e d o n Gonyaulax ce ll s a t t he end of day p has e (c t 12)2.2 A b b re v ia t io n s u sed in t h i s p a p er : c t , c i r c a d i a n t i m e ( c t 0 i s d a w n , c t1 2 d u s k , i n a l i g h t / d a r k c y c l e o f 1 2 h o f li g h t , t h e n 1 2 h o f d a r k n e s s ) ;G T 4 2 9 , a l u m i n o u s s t ra i n o f G o n y a u l a x t a m a r e n s i s ; G T P P , a n o n -l u m i n o u s s t r a in o f G. ta ma ren s i s ; P A S , p e r i o d i c a c i d - S c h i f f s t a i n in g .1436 T.E JOURNAL OF CELL BIOLOGY ' VOLUME lO0, 1985

Gonyaulax ce l l s t hus embedded in aga ros e were obs e rved 1-2 h l a t e r f romb e n e a t h t h e c o v e r s l i p t h r o u g h t h e I M - 3 5 i n v e r t e d m i c r o s co p e . B i o l u m i n e s e e n c ew a s s t im u l a t e d b y a d d i n g a d r o p o f a n i o n s o l u t i o n i n t o t h e w e ll i n d a r k n e s sw h i l e o b s e r v i n g a p r e v i o u s ly f o c u s e d ce ll i n t h e v i d e o m o n i t o r c o u p l e d t o t h eimage- in t ens i f i ed camera an d v ideo t ape recorde r . Becaus e the ions d i f fus et h r o u g h a l a y e r ( - 3 m m ) o f a g a r o s e b e f o r e r e a c h in g t h e c e ll i n f o c u s , h ig hconcent ra t ions were us ed to reduce the t ime requi red to e l i c i t l umines cence : 3M CaC12 or KC I , concen t ra t ed N H4O H, or g l ac i al ace t ic ac id . Mu ch low erconce nt ra t ion s a re s uf f i c i en t for cel ls i n s o lu t io n wh ere mix ing i s rap id ( 14 , 15) .

"S pheroplas t s " l ack ing ce l l wa l l s (1) were prepa red by cent r i fug ing andr e s u s p e n d i n g t h e c el ls i n t h e f o l lo w i n g m e d i u m : 4 0 0 m M N a C I , 5 0 m M K C I ,5 m M M g C I 2 , 1 0 m M N a p h o s p h a t e , 7 .5 m M E D T A , 6 0 m M T r i s , p H 8 .2 .Af t e r 4 -8 h of i ncuba t ion in t h i s med ium , m any ce l l s s hed the i r ce ll wa l ls .Preparat ion of Cel l Extracts; Scint i llon Assay: I l i t e r o f Gon-yautax cul ture w as fi lt e red a t du s k (c t 12) t h ro ugh a W ha t ma n 541 f i lt e r,r e s u s p e n d e d in 5 m l e x t r a ct i o n b u f f e r ( 1 0 0 m M T r i s , 1 0 m M E D T A , 5 m M 2 -merc aptoe th anol , pH 8 . 5 , I C) and th e cel ls were broken by a s ing le pas st h r o u g h a K i r k l a n d e m u l s i f i e r ( B r i n k m a n n I n s t r u m e n t s , I n c ., W e s t b u r y , N Y ) .More than a s ingle ex t rac t ion p as s tended to dec reas e s c in t i l l on ac tiv ity . Ce l ldebr i s was removed by a 10-min cent r i fuga t ion a t 800 g . The pe l l e t was re -ex t rac t ed two or t h ree t imes , each t ime by re s us pens ion in 5 ml of ex t rac t ionb u f f e r w i th a g l a s s h o m o g e n i z e r a n d c e n t r i f u g a t i o n a t 8 0 0 g . T h e s u p e r n a t a n t sw e r e t h e n p o o l e d a n d s p u n a t 2 7 , 0 0 0 g f o r 2 0 r a i n . T h e p e ll et w a s r e su s p e n d e dwi th a g l a ss homo gen ize r i n 2- -4 ml of ex t rac t ion bu f fe r and e i the r l oadeddi rec t ly on to a s uc ros e grad ien t ( s ee be low) or i ncub a ted o ve rn igh t a t I oc int h e d a r k a n d t h e n s u b j e ct e d t o d e n s i t y g r a d ie n t s e d i m e n t a t i o n .

S c in t i l lon ac t iv ity of 10-u l a l iquot s o f ex t rac t was a s s ayed by in j ec t ing acet icac id (12) ; t he in it ia l ma xim um l igh t i n t ens i ty was recorded .

Isodensity Sedimentation: P ar t i a l pur i f i ca t ion of s c in t i l l ons wasa c h i ev e d b y c e n t r i f u g a t io n t h r o u g h s u c r o s e d e n s i ty g r a d i e n t s b y a m o d i f i c a t io nof t he pro cedur e of F oge l e t al . (12). G radien t s were form ed f rom s ix laye rs (5 . 5m l e a c h ) o f 3 5 % , 4 0 % , 4 5 % , 5 0 % , 5 5 % , a n d 6 0 % s u c r o s e a n d s m o o t h e d f o r3 6 - 4 8 h a t 4 C . T h e s u c r o s e so l u t i o n s c o n t a i n e d 5 0 m M T r i s , 1 0 m M E D T A ,a n d 5 m M 2 - m e r c a p t o e t h a n o l a t p H 8 . 1 - o r 2 - m l s a m p l e s o f e x t r a c ts w e r el ayered on to the grad ien t s and s p un for 2 h a t 17 , 000 rpm in an S V-288 ve r t i ca lr o t o r i n a S o r v a l l R C - S B c e n t r i f u g e ( D u P o n t C o m p a n y , N e w t o w n , C T ) ( 0 -1PC). I - t o 1 . 8-ml f rac t ions were co l lec ted and a s sayed . The c once nt ra t ion off luores cent pa r t i c l e s i n t he f rac t ions of s uc ros e grad ien t s was meas ured w i th ah e m a c y t o m e t e r u n d e r t h e f l u o r es c e n c e m i c r o s c o p e ( se e F i g . 6 ). T h e d i s t r ib u t i o no f c h l o r o p la s t s e d i m e n t a t i o n w a s d e t e r m i n e d b y m e a s u r i n g a b s o r p t i o n a t 6 7 5n m i n e a c h o f t h e f r a c t io n s .Recharging of Scintillons In Vitro: R e c h a r g i n g o f s e i n t i l l o n a c ti v-i ty wa s pe r form ed es s en t i a l l y a s des c r ibed b y F oge l and Has t ings ( 11 ) . The peaks e in t i l lon f rac t ion f rom a s uc ros e dens i ty grad ien t was ac id i fi ed to pH 5 . 5 tod i s cha rge i t s s c in t i l lon ac t iv i ty . Th en i t was a lka l in ized b ack to p H 8 an dluc i fe r in pur i f ied a s des c r ibed by F oge l and Has t ings (10) was added . A f t e ri n c u b a t i n g a t 2 0 C a n d / o r I C , t h e f l u o r e s c en t p a r ti c le s w e r e p h o t o g r a p h e d a n dscint i l lon act ivi ty re-assayed.

F l o w C y t o m e t r y : A B e c t o n - D i c k i n s o n F A C S A n a l y z e r ( B e c to n - D i c k -i n s o n F A C S S y s t e m s , S u n n y v a l e , C A ) w a s u s e d t o m e a s u r e t h e e n d o g e n o u sf l u o r es c e n c e o f p o p u l a t i o n s o f i n t a c t Gonyaulax ce ll s . A merc ury l amp p rovidedthe exc i t a t i on l i gh t , which was f i l t e red a s des c r ibed be low. Ce l l s were s lowlyinfus ed throug h a 100-urn or i f i ce us ing mi l l i pore - f il t e red (0 . 22 um ) s eawa te r a sthe s hea th buf fe r . F luores cence in t ens i ty me as ure me nt s f rom a t l eas t 1 , 000Gonyaulax ce l l s were co l l ec t ed w i th a l oga r i thmic ampl i f i e r and the peakpos i t i on of t he re s u l t i ng h i s togram ( i .e . , t he log of f l uores cence in t ens i ty vs .n u m b e r o f c e ll s) w a s e s t i m a t e d b y a B e c t o n - D i c k i n s o n d a t a a n a l y s is p a c k a g e(Cons or t 20 Appl i ca t ion P rogram Vers ion 2 , 1983) . Thi s peak pos i t i on on thelog sca le wa s then conv er t ed to a ca l ib ra t ed l i nea r s ca l e by com par in g the logp e a k p o s i t i o n t o a s t a n d a r d c u r v e g e n e r a t e d b y m e a s u r i n g t h e f l u o re s c e n c e o fc a l ib r a t ed p o l y s t y r e n e b e a d s ( C u r t i s - M a t h e s o n f l u o r e s c e n t " m i c r o s p h e r e s " ) o fd i f fe ren t f l uores cence w i th bo th loga r i thmic and l i nea r ampl i f i e r s by the pro-cedure o f Mui rhead e t a l. (24). Gonyaulax f luores cence a s mea s ured w i th e i the rthe loga r i thmic or l i nea r ampl i f i e r s was equiva l en t a f t e r t h i s convers ion , bu ta c c u m u l a t i o n o f t h e d a t a o b t a i n e d w i t h t h e l o g a r i t h m i c a m p l i f i e r a l l ow e d am o r e p r e c i s e e s ti m a t i o n o f t h e p e a k p o s i t i o n .

The in t e r fe rence f i l t e r s us ed in t he cy tomete r were a s fo l lows : (a ) 350-375exc i t a t ion : 360 _+ 10- nm ban d pas s fi lt e r, 37 5-nm s hor t -pas s f il te r , and 375-n m s h o r t - p a s s d i c h r o i c m i r ro r ; ( b ) 4 7 5 - 4 9 5 e x c i ta t i o n : 4 8 5 + 1 0 - n m b a n d p a s sf i lt e r, 505-nm s hor t -pas s d i chro ic m i r ror ; (c ) 470 -500 emis s ion : 485 + 15- nmband pas s f il te r , 400-n m lon g-pas s f il te r ; (d ) 505-5 45 emis s ion : 525 +- 20 -n mband pas s f il te r ; ( e ) 580-nm long-pas s emis s ion : 580-n m long-pas s f il te r . F i l t e r sw e r e o b t a i n e d f r o m B e c t o n - D i c k i n s o n .

C e l l s w e r e p r e p a r e d f o r f l o w c y t o m e t r y b y p l a c in g 2 - m l a l i q u o t s o f a h e a l th y ,log-phas e cu l ture i n to c l ea r po lys tyren e ti s s ue -cu l ture t ubes (6 ml ) , which werel o o s el y s to p p e r e d . T h e s e a l i q u o t s w e r e d i s p e n s e d o n e d a y o r m o r e b e f o r e t h ee x p e r i m e n t b e g a n f r o m c u l t u r e s w h i c h h a d b e e n s y n c h r o n i z e d b y a l i g h t / d a r k



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at the surface vs periph ery of the cells (Fig. 1 e vs h, and Fig.10a vs b). The appe aranc e of discrete fluorescent particles inth e Gonyaulax cortex is not an artifact due to optical distor-t ions of the cell wall, which had been suggested (33) as anexplanat ion for ear l ier observat ions of compartmental izedbiolumin escenc e (28). For e xample , if cells are gently lysedby squeezing them between cover slip and slide, fluorescent

particles of the app ropriate size and shape are seen in thelysate (Fig. l, i-l), conf i rming that the ex is tence of theseparticles in vivo is not an artifact. This can also be shown byremoving the cell wall; "spheroplasts" display the same fluo-rescent particles (Fig. 1, e-h). Nevertheless, there may besome relationship between the particles and the cell wall .Indeed, the fluorescent particles o f cells with intac t walls often

FIGURE 2 Endogenous flu ores cence of G. tamarensis. (a-c) Two G. tamarensis cells, strain GT429 (luminous). (d- f ) Two G.tamarensis cells, strain GTPP (nonluminous). (a and d, EX 395-4 20; EM 450-560 ; b and e, EX 450 -490 ; EM 520-560 ; c and f,br ight f ield. Tr i-X f i lm w ith Acuf ine developmen t.) 40x Ne ofluor object ive.1438 THE JOURNAL OF CELL BIOLOGY VOLUME I0 0, 1985



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FIGURE 4 Co- local izat ion of endo genous f luorescence w i th b io luminescence st imulated by NH4 +, Legend and procedure as inFig. 3, except that exposure times for A and C are comparable.

found that the intensi ty of the endogenous f luorescence isdiminished after the flash but remains in the same loci (Fig.3 C) , as demon strated here in an overexposed pr int o f thispanel. Finally, the bright-field picture in Fig. 3D is includedto orien t the reader as to the size and p osition o f the cell.Fig. 4 illustrates the same experimental protocol performedupon a cell stimulated by NH4 + (in this case, Fig. 4, A and C,were photographed with comparable exposures) . Again, thebioluminescence and fluorescence are co-localized. Similarresults were obta ined w ith high K and acetic acid (data n otshown).The fact that the particulate f luorescence is considerablydiminished after exhaustive chemical stimulation (Fig. 3A vsC; Fig. 4A vs C) supports the interpretation that luciferin isthe fluorescent entity: the fluorescence o f dinoflagellate luci-ferin is known to be lost in vitro after oxidation by luciferase(2) or autoxidation by air (8). Therefore, the in vivo fluores-cence data cor roborates the in vi tro behavior o f lucifer in.We were able to measure the kinet ics of the fash f rom asingle particle by using a video analyzer attached to a str ip-char t recorder (see Mater ials and M ethods) . The durat ion ofbioluminescent f lashes in vivo and in vi tro has been measuredto be ~ 100 ms (19), whereas the flash duration illustrated inFig. 5 is -300 ms. This discrepancy is probably due either tothe lag inherent in the video camera and/or to the dif ferentmeth ods used to stim ulate the cells ( i.e. , chemical vs mecha n-ical stimulation). Nonetheless, the f lash kinetics of the video1440 TH[ JOURNAL OF CELL BIOLOGY - VOLUME 100, 1985

record are similar to those o f a biolu mine scent f lash. Fig. 5also illustrates the decrease of endogenous fluorescence sub-sequent t o the flash by the difference in the baseline (fluores-cence under dim excitation) before and after the flash. Occa-s ionally we have observed mult iple f lashes f rom im mobil izedcells after ionic stimulation. When this happens, it appearsthat most of the particles contribute to each flash. Therefore,mult iple f lashes f rom a cel l are not due to recrui tment of adifferent population of particles for each flash, but eachindividual particle can emit multiple f lashes until the biolu-minesce nce capa city of the particle is exhausted, as is alsotrue for Noctilucamicrosou rces (9).Autofluorescent Particles In Vitro

Previous work had characterized the scintillon as a self-contained sedimentable par t ic le that em it ted a l ight f lash uponrapid acidification (19). When Gonyaulaxextracts were cen-trifuged into a sucrose gradient, the scintillon activity co-sedime nts with the fluorescent particles (Fig. 6). This experi-ment supports the hypothesis that these fluorescent particles,which also emi t the biolumi nescen t f lash in vivo, are identicalwith the previously described scintillons.Ano ther characteristic shared by scintillons and the fluores-cent particles is their ability to be recharged by luciferin atpH 8. Fogel and Hastings (11) discovered that scintillonswhose act ivi ty had been discharged by acidif icat ion could be



7/12

q~

I I I I I I I I0 2 4 6 8seconds

F IG UR E 5 K i n e t i c s o f t h e b i o l u m i n e s c e n t f la s h fr o m a s in g l e m i c r o -s o u r c e ( s c i n t i l l o n ) i n v i v o ( s e e M a t e r i a l s a n d M e t h o d s ) . T h i s c e l l w a se x c i t e d w i t h v e r y d i m l i g h t (E X 3 9 5 - 4 2 0 ) a n d t h e b i o l u m i n e s c e n tf la s h s t i m u l a t e d b y C a + + ( 2 0 C ) . T h e r e f o r e , t h e b a s e l i n e i n t e n s i t yb e f o r e t h e f l as h is d u e t o d i m f l u o r e s c e n c e u p o n w h i c h t h e f la s h iss u p e r i m p o s e d . N o t e t h a t , a f t e r t h e f l as h , th e i n t e n s i ty o f t h e e n d o g -e n o u s f l u o r e s c e n c e h a s d e c r e a s e d .

5 . I15 3

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C o - s e d i m e n t a t i o n o f f l u o r e s c e n t p a r t ic l e s a n d s c i n t i ll o na c t i v i t y i n s u c r o s e g r a d i e n t s . S c i n t i l l o n a c t i v i t y ( O ) ; c o n c e n t r a t i o n o ff l u o r e s c e n t p a r t i c l e s ( 0 ) ; c h l o r o p l a s t s ( A ) .

r e c h a rg e d b y in c u b a t io n w i th p u r i f i e d lu c i f e r in a t p H 8 . Wefind tha t the f luorescence o f pa r t ic les d ischarged in v i t ro canbe s im ila r ly res to red . F ig. 7 shows f luoresce n t pa r t ic les in v i t roth a t c a n b e d i s tin g u i s he d f ro m o th e r f lu o re s c e n t m a te r i a l b ythe same c r i te r ion used with in tac t ce l ls , namely f luorescencetha t is p resen t under 395-420-nm exc i ta t ion (F ig . 7 , b , d , andf ) bu t no t 45 0-4 90 -nm exc i ta t ion (F ig. 7 , a , c , and e ) . F ig. 7 bshows two f luo rescen t pa r t ic les in v i t ro (a rrows) . Ac id if ica t ioncon com itan t ly d ischarges the f luorescence (F ig . 7 d ) an d sc in -t i ll o n a c t iv i ty. B u t a f t e r r e a d ju st in g th e p H to 8 a n d in c u b a t in gwith luc ife r in , the sc in t i l lon ac t iv i ty was res to red to 45% ofthe o r ig ina l leve l and f luorescen t pa r t ic les reappeared in thee x t ra c t ( tw o s h o w n b y a r ro w s in F ig . 7 f ) . In a d d i t io n tofu r th e r s u p p o r t in g th e n o t io n th a t f l u o re s ce n t p a r ti c l e s a re th es a m e a s s c in ti l lo n s , t h i s e x p e r im e n t c o n s t i t u t e s t h e s t ro n g e stev idence tha t the f luorescence o f these pa r t ic les is tha t o fluc ife r in tha t i s bound to the pa r t ic les a t a lka l ine pH.Flow C ytome tric Studies

W e e m p l o y e d f l o w c y t o m e t r y t o q u a n t i f y t h e c h a n ge o fendo geno us f luorescen ce in in tac t ce l ls (a ) a f te r the d ischargeo f b io lu m in e s c e n c e a n d (b ) o v e r t h e c o u r s e o f t h e c i r c a d ia n

cyc le . A s t r ic t l inea r correspondence be tween ce l lu la r f luores -c e n c e a n d lu c i fe r in c o n te n t m ig h t n o t b e p o s s ib l e b e c a u s e o fth e o p t i c a l p ro p e r t i e s o f t h e c e ll s a n d th e p o s s ib l e q u e n c h in gof luc ife r in f luorescence in the pa r t ic les (sc in t i llons ) . Nev er-theles s , mea suring ce l l f luorescence exc i ted by l igh t o f thea p p ro p r i a t e w a v e le n g th s h o u ld g iv e d a ta th a t r e f l e c t s t h eluc ife r in concen tra t ion in v ivo .

F ig. 8 i l lus t ra tes " sca t te r p lo ts " o f ce l l vo l um e vs f luores -c e n c e fo r Gonyaulaxcu l tu res . F ig . 8 , a and b , a re da ta f romc u l tu re s w h o s e f lu o re s c e n t e m is s io n w a s m o n i to re d a t 4 7 0 -5 0 0 n m a f t e r e x c i t a t io n a t 3 5 0 -3 7 5 n m (a c o m b in a t io n th a tis com para b le to the m ic rog raph s o f F igs . 1 , a , e , and h , and2 a ) . T w o f lu o re s ce n t p o p u la t io n s a re o b s e rv e d in F ig. 8 a : o n eg ro u p o f s m a l l v o lu m e a n d lo w f lu o re s c en c e a n d a n o th e rg ro u p o f la rg e r v o lu m e a n d m u c h g re a t e r f l u o re s c e nc e . T h ela t t e r g ro u p i s Gonyaulax, a s c a n b e d e m o n s t ra t e d b y f i l te r ingthe cu l tu re th rough a 25-#m Nitex f i l te r , which t raps Gonyau-lax c e ll s ( ce ll d i a m e te r , - 3 5 # m ) ; o n ly th e s m a l l e r v o lu m e p e rf lu o re s c e n c e p o p u la t io n r e m a in s . We b e l i e v e th e s e s m a l l e rf lu o re sc e n t p a r ti c l e s t o b e th e b a c te r i a t h a t c o n ta m in a te o u rGonyaulax c u l tu re s . F o r o u r f lo w c y to m e t ry e x p e r im e n t s , w ee x c lu d e d th e s ig n a l f ro m th e b a c te r i a b y s im p ly r a i s in g th et r ig g e r in g th re s h o ld fo r c e l l v o lu m e u n t i l n o b a c te r i a w e rerecorded , a s in F ig . 8 b . W e were the reby ab le to se lec t ive lym e a s u r e Gonyaulax f luorescence in our s tandard cu l tu res ,and th is is the way the da ta in F igs . 9 and 11 and Tab le Iwere co l lec ted .F ig . 8 , c a n d d , s h o w tw o o th e r c o n t ro l m e a s u re m e n t s . T h ece l ls in F ig . 8c were exc i ted with 475-495-nm l igh t andf lu o res c e n c e e m is s io n m o n i to re d a t 5 0 5 -5 4 5 n m (c o m p a ra b leto the m ic rog raph s o f F igs . l , b an d f , and 2 , b and e ) . Aga in ,b o th th e b a c te r i a (i .e ., t h e c lu s t e r o f m a n y s m a l l e r v o lu m ecells) and Gonyaulax ( i . e . , the few la rge vo lume ce l ls abovethe bac te r ia l c lus te r) a re f luorescen t , bu t the ir in tens i t ie s a reno t ve ry d iffe ren t . Th is is cons is ten t wi th the f ind ing tha tGonyaulax ce l ls have neg l ig ib le pa r t icu la te f luorescence i fexc i ted a t 450 -490 nm (F igs . l , b and f , and 2 , b and e ) , andin d ic a te s th a t t h e f lu o re sc e n c e m e a s u re d b y f lo w c y to m e t ry i sno t an a r t i fac t o f , fo r exam ple , l igh t-sca t te r ing . F ig . 8 d dep ic tsth e v o lu m e / f lu o re s c e n c e p lo t s fo r a c u ltu re e x c i t e d b y 3 5 0 -3 7 5 -n m l ig h t, b u t m o n i to re d fo r f lu o re s c e n ce a t w a v e le n g th s>5 8 0 n m . T h i s f i lt e r c o m b in a t io n a l lo w s c h lo r o p h y l f lu o re s-c e n c e o f Gonyaulax to be recorded . As expec ted , the la rge rd inoflage l la te ce l ls d isp lay more red f luorescence than thes m a l l e r b a c te r i a t h a t d o n o t h a v e c h lo ro p h y l l . T h e s e o b s e r -v a t io n s c o n f i rm th a t t h e c e l l f l u o re s c e n c e m e a s u re d in F ig .8 b is tha t o f Gonyaulax.T o q u a n t i fy a u to f lu o re s c e n c e w i th th e f lo w c y to m e te r , w eco l lec ted the da ta f rom 1 ,000 Gonyaulax ce l ls wi th the am-p l i f i e r i n a l o g a r i th m ic m o d e a n d a r ra n g e d in to a h i s to g ra m(Fig. 9 ) . T h e p e a k o f t h is h i s to g ra m a s e s t im a te d b y th e d a taa n a ly s i s p ro g ra m w a s th e n c o m p a re d to a c a l ib ra t io n c u rv ec a lc u lat e d b y m e a s u r in g th e r e l a t iv e f lu o re s ce n c e o f p o ly s ty -re n e b e a d s o f c a l ib ra t e d f lu o re sc e n c e in t e n si t i es b y th e p ro -cedure o f M uirh ead e t a l . (24) . Th is " re la t ive f luorescence " isthe quan t i ty used in Tab le I and F ig. 11 .T a b le I s h o w s th a t s t im u la to ry t r e a tm e n t s t h a t e x h a u s tb io lu m in e s c e n c e w i l l d i sc h a rg e m o s t o f t h e a u to f lu o re s c e n c eas we l l. T h is e ffec t i s pa r t icu la r ly s ign if ican t fo r ce l ls in the irs u b je c tiv e n ig h t p h a s e , w h e n th e b io lu m in e s c e n c e c a p a c i ty isg rea tes t (and a lso au to f luorescence ; see be low). Hydrogen ,c a l c i u m , p o t a s s i u m , o r a m m o n i u m i o n s a t c o n c e n t r a t i o n stha t d ischarge b io luminescence a lso reduce ce l lu la r f luores -JOHNSON ET AL. Compartmenta l i za ti on o f A lga l B io luminescence 1441



8/12

1442 THE JOURNAL OF CELL BIOLOGY VOLUME 100, 198 5



9/12

F~GURE 8 Flow cyto me ter scatter plots of cell volu me (VOL) vs fluorescence (FL) of Gonyaulax polyedra cultures, a and b (EX350-3 75; EM 470-500), scintil lon fluorescence , b is the same as a except that triggering thresh old for volum e has been raised toexc lude th e bacteria, c (EX 475 -495 ; EM 505-54 5), PAS-body fluoresc ence; the vo lume gain in this panel is slightly less than forthe oth er three panels, result ing in an app arent ly smalier votume for the Gonyaulax ceils, while the fluorescence gain has beenadjusted to com pensate for the dif ference in e xcitat ion intensity betwe en EX 350-375 and EX 475-495 as can be seen from thefact that the ba cterial fluorescence in a and c are comp arable , d (EX 350-3 75; EM 580 long pass), chlo roph yll fluorescence. Thetriggering thresh old for volu me is higher in c and d than in a, causing a "chop ped off" d istributio n. Both volum e and fluorescencescales are log arithm ic scales.

cence by more than half, whereas equivalent concentrat ionsof sodium ions, which do not tr igger bioluminescence (15),do not af fect Gonyaulax f luorescence. This result is compa-rable to that v isualized in Figs. 3 and 4.Since bioluminescence in vivo is s t imulated by mechan icalagita tion (35) , we were concerned that the m easuremen ts oflucifer in f luorescence might be com promis ed by s t imulat ionof the cel ls to em it biolum inescence dur ing their passagethroug h the orif ice of the cytomete r. This might differentiallyalter the amount of luciferin f luorescence at different timesof day, because Gonyau[axcells are more sensitive to me-chanical s t imulat ion dur ing the night phase (5) . We circum-vented this poss ible problem by us ing cel ls that had been

"photoinhibi ted" by the exposure to continuous dim i l lumi-nation; such cells are far less susceptible to mechanical stim-ulation o f biolum inescen ce (4, 36). Prelim inary results notincluded here conf irmed the predict ion that the autof luores-cence of night phase cells as measured by f low cytometry islower in nonpho toinhibi ted cel ls as compared with cel ls thathave been pho toinhibi ted as in the fol lowing exper iments .Table I includes data showing that ionic t reatments haveless effect upon the fluorescence of cells in th eir subjective"day" phase, as is also true for ionic effects on biolumines-cence (14). Ta ble I also shows that u nstim ulate d cells in theirday ph ase are substantially less f luorescent than those in nightphase as is evident as well from the histogram plots of Fig. 9.

FIGURE 7 Restoration of fluores cence of discharged particles in vitro by luciferin, a and b, c and d, and e and f are representative,but n ot ide ntical fields, a (EX 450-4 90) and b (EX 395-420 ), con trol particles in vitro (arrows) before discharge by acidifica tion, c(EX 450 -490 ) an d d (EX 395-420), disappe arance of fluorescen t particles after acid discharge (pH read justed 1~o8.0 for micrograph).e (EX 450-4 90) and f (EX 395-420), reappea rance of fluorescent particles (arrows) after incub ation of same extract with luciferinat pH 8 (incubated for 1.5 h at 20C, then 2 h at 0C). Fluorescence that appears und er both EX 450 -490 and EX 395 -420 ismost ly that of chloroplasts, whi le that visualized only und er EX 395 -420 is that of the scintil lons. Tri-X film w~th Acufinedevelopment. 100x object ive.

JOHNSON Er AL Compartmentalization f Algal Biolurninescence 1443



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day night

t~

0 5 0 I 0 0f l uorescence

F IG U RE 9 H i s t o g r a m p l o t s o f G . polyedra f l u o r e s c e n c e f r o m c e l l sa t d a y a n d n i g h t p h a s e i n c o n s t a n t l ig h t . O r d i n a t e is "c e l ls p e rc h a n n e l " i n r e la t i v e li n e a r u n it s , w h i l e " c h a n n e l " o n t h e a b s c i ss ar e f e r s t o a g i v e n f l u o r e s c e n c e i n t e n s i t y m e a s u r e d b y t h e c y t o m e t e r ' sa m p l i f i e r i n t h e l o g a r i t h m i c m o d e . 1 , 0 0 4 ce l ls c o n t r i b u t e d t o t h e" d a y " h i s t o g r a m , w h i l e t h e " n i g h t " h i s t o g r a m i s t h e d i s t r i b u t i o n o f9 9 8 c e l ls . T h is e x p e r i m e n t i s t h e s a m e a s t h e d a y a n d n i g h t c o n t r o l si n E x p e r i m e n t 2 o f T a b l e I .

TABLE IEffect o f D i f ferent Treatments on Fluorescence of "N igh t" a nd"D a y" Phase C e l l s in C ons tan t L ight

R e l a t i v e f l u o r e s c e n c eT r e a t m e n t * E x p t . # 1 il E x p t . # 2 ~

N i g h t c e l l s (ct 17)C o n t r o l 1 0 0 1 0 0N a + 1 0 0 9 8K + 5 2 3 3N H 4 + 4 4 4 2A c i d 4 4 2 9C a + + 4 7 4 2Day ce l ls (c t 7 )C o n t r o l 3 8 3 8N a 3 9 3 8K 2 8 2 7N H + + 3 0 2 7A c i d 3 3 2 9C a + + 3 4 3 1

c t , c i r c ad ian t ime ." F i nal c onc en t ra t i ons o f i on i c t rea tmen t : 150 mM NaCI , 150 mM KCI , 100m M N H + C I , 3 3 m M a c e t i c a c i d o r 1 5 0 m M C a C I 2. C e l l f lu o r e s c e n c e w a sm e a s u r e d - 1 0 s a ft e r th e i o n i c s t i m u l a t io n .I I Same ex pe r ime n t as dep i c ted i n F i g . 11 . S a m e e x p e r i m e n t a s d e p i c t e d i n F i g . 9 .The da i ly va r ia t io n in ce l lu la r f luorescence is eas i ly v isua l izedin F ig . 10 , which c on tras ts the in tens i ty o f pa r t icu la te f luores -cence f rom ce l ls in the sub jec t ive n igh t vs day , which werefocused at the cell surface (Fig. 10, a and c) and the cellp e r ip h e ry (m e d ia n o p t i c a l se c t io n ) , w h e re th e n ig h t /d a y d i f -fe rence is m os t o bv iou s (F ig . lO, b and d ) . A s seen in F ig . lO,a a n d c , t h e n u m b e r o f p a r t ic l e s p e r c e ll a p p e a r s t o r e m a inra th e r c o n s t a n t t h ro u g h o u t t h e c i r c a d ia n c y c le , w h e re a s th e i r1444 THE JOURNAL OF CE LL BIOLOGY . VOLU ME 100, 1985

i n t e n s i ty c h a n g e s . T h e d a i ly m o d u la t io n o f Gonyaulax luo-rescence corre la tes wi th tha t o f ex trac tab le luc ife r in (4 ).

T h i s d a i ly c h a n g e o f a u to f lu o re s c e n c e i s co n t ro l l e d b y th eendogenous c i rcad ian c lock , a s i l lus t ra ted in F ig . 11 . Th ise x p e r im e n t s h o w s th a t Gonyaulax l u o re sc e n c e i s rh y th m i -c a l ly m o d u la t e d a n d p e r s is t s fo r a t l e a st 4 8 h u n d e r c o n s t a n te n v i ro n m e n ta l c o n d i t io n s . T h e t im e s o f g re a t es t f lu o re s c en c eco inc ide with g rea tes t capac i ty to b io luminesce (sub jec t iven igh t , CT 12 corresponds with dusk) . Ca lc ium d ischargesf lu o res c e n c e to a b a c k g ro u n d l e v el w h ic h m ig h t i t s e l f b erh y th m ic (b u t i f s o, i t s a m p l i tu d e i s v e ry s m a ll ) . T h e m a g n i -tude o f the osc i l la t ion in to ta l ce l l f luorescence is a t leas t 2 .5 -fo ld ; i f th e b a c k g ro u n d d e f in e d b y th e f lu o res c e n c e r e m a in in ga f t e r C a ++ d i s c ha rg e i s s u b t ra c te d , t h e rh y th m ' s a m p l i tu d em a y b e a s m u c h a s 2 5 -fo ld. T h u s , t h e c i r c a d ia n c lo c k d i r ec t ss u b s t a n t i a l c h a n g e s in lu c i f e r in c o n te n t a s c a n b e m e a s u re db y th e f lo w c y to m e te r in v iv o .DISCUSSIONCom partmental izat ion o fBioluminescence Compo nents

B io lu m in e s c e n c e o f Gonyaulaxo r ig in a te s f ro m s u b c e l lu l a rpa r t ic les tha t a re f luorescen t . These pa r t ic les appear to be thes a m e a s th e in v i t ro s c in t i ll o n s b e c a u s e th e y c o - s e d im e n t i ns u c ros e g ra d ie n t s a n d h a v e th e s a m e c h a ra c te r i s ti c s o f r e c h a rg -ing by luc ife r in in v i t ro . Con sequen t ly , sc in t i l lons do no t seemto be an a r t i fac t o f ex trac t ion , e spec ia l ly s ince the appear anceo f th e f lu o re s c e n t p a r t i cl e s i n v i t ro m a tc h e s th a t i n v iv o .Th e f luorescence o f these pa r t ic les can be a t t r ibu ted toluc ife r in because (a ) the ir f luorescence exc i ta t ion /emiss ioncharac te r is t ic s a re comparab le to luc ife r in , (b ) the ir f luores -cence is los t a f te r exhaus t ive b io luminescen t emiss ion , a s isthe f luorescence o f luc ife r in , (c ) the f luorescence in tens i tyundergoes a da i ly osc i l la t ion (as does luc ife r in ) , and (d ) thef luorescence o f d ischarged pa r t ic les can b e res to red in v i t rob y a d d in g lu c if e r in a t p H 8 . T h e re fo re , a s j u d g e d b y i t sf luorescence , the vas t m a jor i t y o f luc ife r in is a s soc ia ted withthese cor t ica l pa r t ic les in v ivo . Th is obse rva t ion impl ies tha tluc ife r in b ind ing p ro te in (32) is a lso as soc ia ted with the pa r-t ic les (20) . As with many chromophores , the f luorescenceq u a n tu m y ie ld o f l u ci f er in m a y b e d i f f e re n t w h e n p ro te inb o u n d (3) , a n d th i s e f f e ct m a y d i f f e r d e p e n d in g o n th e p ro te in .Un l ike iuc ife r in , the in t race l lu la r d is t r ibu t io n o f the b io lu -m in e s c e n c e e n z y m e lu c i f e ra s e i s u n c le a r . S o m e p o r t io n o fluc ife rase is de f in i te ly as soc ia ted with sc in t i l lons (20) , bu t thereported re la t ive p rop ort io n o f luc ife rase in the sc in t i l lon vsso lub le poo ls va r ies cons ide rab ly (19, 23 , 37). Th is rep ortedvar iab i l i ty ce r ta in ly de r ives in pa r t f ro m d iffe rences in ex trac -t io n p ro c e d u re s a n d th e t im e o f t h e c i r c a d ia n c y cle c h o s e n fo rex trac t ion , bu t i t has been d iff icu l t to e l imina te ex trac t iona r t i fa c t s w h e n th e o n ly a s s ay o f t h e e n z y m e ' s lo c a l i za t io n h a sbeen i ts ac t iv i ty in v i t ro . Reso lu t ion o f th is is sue m ay hav e tow a i t fo r im m u n o c y to c h e m is t ry o f i u ci f e ra s e a t t h e u l t r a s tru c -ru ra l le v el . In th e m e a n t im e , i t i s im p o r t a n t t o n o te th a t t h elo c a li z a t io n o f a s in g le c o m p o n e n t o f t h e b io lu m in e s c e n c ere a c t io n - -n a m e ly , l u c i f e r in - -w i l l r e s u l t i n l o c a l i z e d b io lu -minescen t emiss ion i r respec t ive o f whe ther luc ife rase is d is -t r ib u te d th ro u g h o u t t h e c y to p la s m o r i s r e s t r i c t e d to th esc in t i l lons in v ivo .Ce llular P hysiology of Co nyau lax Flashing

F o g e l a n d H a s t in g s (1 1 ) h a v e s u g g e st e d th a t t h e m e c h a n i s mof sc in t i llon f la sh ing invo lves a loca l ac id if ica t ion tha t bo th



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FIGURE 10 Endog enous fluore scen ce of C. polyedra cells in night and da y phases of the circadian cycle (all photos: FX 395-420;EM 450-560). Cells previously synchronized in a light/dark cycle were placed in continuous dim light at dawn (ct 0). a (surfacefocus) and b (peripheral focus) are photos of night-phase cells after 18 h in dim light (ct 18). c (surface focus) and d (peripheralfocus) are photos of day-phase cells after 27 h in dim light (ct 3). Ektachrome 160 film. 40x Neoflu or objective.

FIGURE | 1 Circadian variation in endoge nous fluorescence o f G.polyedra as measured by flow cytometry. Cells previously synchro-nized in a l ight/dark cycle were placed in continuous dim light (LL)at dusk (ct 12). The relative fluorescence (EX 350-375; EM 470500) of cells at 1-h intervals was measured before and after thedischarge of biolum inescenc e with 150 mM CaCI2. This exper imentwas perfo rmed at 25C and is the same as Experiment 1 in Table I.releases luciferin from its binding protein and activates lucif-erase, resulting in light emission. That protons are the activeagent is indicated b y the kinetic similarity of flashes in vivo

and those elic ited in vi tro by a rapid pH drop and by the factthat no other ion provokes l ight emiss ion f rom scint i l lons(l 9). A m ore specific mod el based o n the properties in vitroof luciferase and luciferin binding protein (20) was laterpropo sed (18).How might such a proton f lux result f rom mechanicalstimula tion (shear) of the cell surface? H am m an and Seliger(14, 15) have propo sed that turbul ence causes a depolarizationof the plasma m em br an e-- a s ignal that is ul timately trans-duced into the acidif icat ion. They marshal evidence thatcalcium ions are the depolar izing cat ion, while the subsequentsteps in the tr iggering pathway are unclear. However, sinceneither calcium ions or Ca++/calmodulin will evoke a f lashfrom scintillons in vitro (C. H. Johnson, unpublished obser-vations), unidentif ied intermediate steps must exist.

In the cases of the biolum inesce nt dinoflagellates Noctilucaand Pyrocystis, stimulation depolarizes the tonoplast, whichappears to al low a proton f lux f rom the central vacuole intothe peripheral cytoplasm (25, 38). This cytoplasmic acidifi-cat ion prob ably tr iggers the f lash f rom the luminescent micro-sources (9, 39) by the same m echanism proposed for Gonyau-la x (1 l).Although i t is tempting to conclude that the scenar io forflash stimulation will be the same for Gonyaulax as forNoctiluca and Pyrocystis, the o rganization of these cells isJOHNSON ET AL. Compartmentalization f Algal Bioluminescence 1 445



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qui t e d i f f erent . I n part i cu lar , Noctilucaa n d Pyrocystish a v e al a rg e c e n t r a l v a c u o l e , w h e r e a s Gonyaulaxh a s a m u c h s m a l le rp e r ip h e r a l v a c u o l e o f u n k n o w n a c i d it y . I n n o n e o f t h e se c e l lsh a s t h e p r o x i m i t y o f s c i n t il l o n s / m i c r o s o u r c e s t o v a c u o l e s( w h i c h a r e t h e p u t a t i v e s o u r c e o f a c i d ) b e e n c a r e fu l ly d o c u -m e n t e d . O n e a l t e r n a ti v e t o t h e Noctilucam o d e l w o u l d b e t h a tGonyau/ax s c i n ti l lo n s m i g h t b e m e m b r a n o u s v e s i c le s e n c l o s -i n g a n a c i d i c m i l i e u t h a t i n v i v o i s t h e p r o t o n s o u r c e t h a tt ri gg e rs l u m i n e s c e n c e . T h e r e f o r e , c a u t i o n i s a d v i s e d a g a i n s t ab r a s h a m a l g a m a t i o n o f t h e e v i d e n c e f r o m t h e s e d i s p a r a t ed i n o f l a g e l l a te s . U n d o u b t e d l y , u l t r a st r u c t u r a l i d e n t i f i c a t i o n o fGonyaulax s c i n t i l l o n s a n d t h e i r r e l a t i o n s h i p t o o t h e r o r g a n -e l l e s w i l l h e l p r e f i n e o u r h y p o t h e s e s c o n c e r n i n g t h e c e l l u l a rp h y s i o l o g y o f f l a s h in g .Clock Co ntrol of Bioluminescence

T h e c a p a c i t y o f Gonyaulax c e l l s t o e m i t l i g h t is a f u n c t i o no f t h e t i m e o f d a y; t h e e n d o g e n o u s c i r c a d i a n c l o c k c o n t r o l st h i s m o d u l a t i o n ( 1 7 ) . U n d e r s t a n d i n g t h e m e a n s b y w h i c h t h ec l o c k r e g u l a te s th i s " d r iv e n " p h e n o m e n o n i s a m a j o r o b j e c t iv eo f o u r s t r a te g y t o u n c o v e r t h e m e c h a n i s m o f t h e c l o c k i ts el f.I n t h e c a se o f b i o l u m i n e s c e n c e c a p a c it y , th e c l o c k c h a n g e st h e i n t r ac e l lu l a r c o n t e n t o f e a c h c o m p o n e n t o f t h e r e a c t io n :l u c i f e r a s e ( 2 2 ) , l u c i f e r i n ( t h i s p a p e r a n d r e f e r e n c e 4 ) , a n dp r o b a b l y l u c i f e r i n b i n d i n g p r o t e i n a s w e l l ( 3 2 ) . A l t h o u g h s u c ha m e c h a n i s m s e e m s w a s t e f u l , i t m i g h t b e t h a t f o r c i r c a d i a nc l o c k s - - a c e l l u l ar e v e n t w i t h a r e l at i ve l y l o n g t i m e - c o n s t a n t - -t u rn o v er o f c o m p o n e n t s m a y p r o v e t o b e a c o m m o n m o d eo f r e g u l a t i o n ( 2 2 ) .W e t h a n k Z e i s s I n c . f o r m a k i n g t h e Z e i s s / V e n u s c a m e r a a v a i l a b l e t ou s , D r . D i e t e r R e c k t e n w a l d o f B e c t o n - D i c k i n s o n f o r t h e C o n s o r t 2 0p r o g r a m a n d D r . A l e x S a u n d e r s f o r t h e s t a n d a r d f l u o r e s c e n t b e a d s .W e a r e g r a te f u l to P r o f e s s o r S a m L a t t f or m a k i n g t h e f l o w c y t o m e t e ra v a i l ab l e t o u s a n d M a r c L a l a n d e a n d R o b e r t H o f m a n n f o r a s s i st a n c ew i t h i t s o p e r a t i o n . W e t h a n k J o h n L u p o f o r p h o t o g r a p h i c a d v i c e ,D o u g L u t z , M a r i e - T h e r e s e N i c o l a s , a n d J a m e s R o e b e r f o r m i s c e l l a -n e o u s b u t i n v a l u a b l e h e l p .P r e l i m i n a r y r e p o r t s o f t h i s s t u d y h a v e b e e n p u b l i s h e d i n a b s t ra c tf o r m : Biophys. J . 4 5 : 2 2 0 a ( 1 9 8 4 ) a n d J . Cell Biol. 9 9 : 5 8 a ( 1 9 8 4 ) . T h i sr e s e ar c h w a s s u p p o r t e d i n p a r t b y g r a n t s f r o m t h e N a t i o n a l I n s t i t u t e so f H e a l th ( G M 1 9 5 3 6 t o J. W . H a s t i n g s a n d G M 3 1 6 1 7 t o S . I n o u r)a n d t h e N a t i o n a l S ci e n c e F o u n d a t i o n ( P C M 8 3 0 9 4 1 4 t o J . W . H a s -t i n g s a n d P C M 8 2 1 6 3 0 1 t o S . I n o u r ) .Received for publication 25 September 1984, and in revised form 13November 1984.

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