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Journal of Muscle Research and Cell Motility 4, 253-262 (1983)

Pyrene actin" do cu m en tat io n of the validity o f asensit ive assay for actin polymerizationJ O H N A . C O O P E R , S I M O N B. W A L K E R a n dT H O M A S D . P O L L A R DDepartment of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore,Maryland 21205, U.S.A.Received 13 September 1982 and in revised form 26 November 1982

SummaryThe fluorescence of pyrene-labelled actin is much higher after polymerization. We havecharacterized in detail the polymerization properties of pyrene actin and report that native andpyrene actin are identical using the following criteria: (1) the time course of polymerization; (2) theelongation rate constants; (3) the intrinsic viscosity; and (4) the critical concentration. Native andpyrene actin copolymerize. Fluorescence of polymerized pyrene actin is 7-10 times higher thanmonomer. The fluorescent signal is proportional to polymer weight concentration and isinsensitive to filament length distribution. Bleaching can be minimized by appropriate filters toallow continuous monitoring of signal. Measurements do not influence polymerization kinetics.This establishes that pyrene actin fluorescence is a valid assay for actin polymerization that is moresensitive than any other current assay.

Introduct ionFluorescence assays of actin polymerization are highly useful because the signal isgenerally proportional only to polymer weight concentration and the sample need notbe disturbed by flow. Other commonly employed assays, such as viscosity and flowbirefringence, are sensitive to polymer length distribution but subject the sample toshear, which breaks filaments. In 1981, Kouyama & Mihashi reported the preparationof pyrene actin by the reaction of actin with N-(1-pyrenyl)iod oacetamide, a thiol-specificreagent. The stoichiometry was 1.0 mol mol ~, and the pyrene was p robably located onCys 374, under the new assignments (Korn, 1982), the fast reacting of the five Cysresidues of muscle actin. At appropriate wavelengths the fluorescence of the polymerwas 20 times higher than the monomer. This was an important finding because otherassays which do not disturb the sample, such as OD 232, light scattering and otherfluorescent probes have limited sensitivity (reviewed in Cooper & Pollard, 1982).

Before a modified protein, such as pyrene actin, can be used as a probe in a detailed0142-4319/83 $03.00 + .12 1983 Chapman and Hall Ltd.

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2 54 C O O P E R , W A L K E R a n d P O L L A R Bq u a n t i t a t iv e a n a l y s i s o f t h e p r o p e r t i e s o f t h e n a t i v e p r o t e i n , i t m u s t b e r i g o r o u s l y e s t a b -l i s h e d t h a t th e p r o p e r t i e s a r e n o t a l te r e d b y th e C o v a l e n t m o d i f i c a t i o n . P r e v i o u s l y ,K o u y a m a & M i h a s h i ( 1 9 8 1 ) m e a s u r e d t h e cr it ic a l c o n c e n t r a t i o n o f p y r e n e a c t in . T h e i rr e s u l t w a s a r e a s o n a b l e v a l u e . A l t h o u g h p r o m i s i n g , t h is e x p e r i m e n t w a s n o t s u ff ic i en tt o e s t a b l i s h t h e v a l i d i t y o f p y r e n e a c t in a s a n a s s a y f o r a c t in p o l y m e r i z a t i o n , s o w e h a v ep e r f o r m e d a d d i t i o n a l e x p e r i m e n t s to c h a r a c t e r i z e p y r e n e a c ti n . I n d e p e n d e n t l y , T e l l a m& F r i e d e n ( 1 9 8 2 ) h a v e c a r r ie d o u t v a r i a t io n s o f t w o o f t h e e x p e r i m e n t s w h i c h w ed e s c r i b e h e r e .

M a t e r ia l s a n d m e t h o d sPreparation of pyrene actinW e m a d e s e v e r a l m i n o r m o d i f i c a ti o n s in t h e p r o c e d u r e o f K o u y a m a & M i h a s h i ( 1 98 1) . W ep r e p a r e d a c h l o r o f o r m - w a s h e d a c e t o n e p o w d e r o f r a b b i t s k e le t a l m u s c l e a n d e x t r a c t e d th ep o w d e r w i t h B u f f e r A ( 2 m M T r i s -H C 1 , p H 8 .0 , 0 . 2 m M C a C 1 2 , 0 . 2 r a M A T P , 0 . 5 r a Md i t h i o th r e i t o l) . T h e ac t i n w as p o l y m er i ze d an d co l lec t ed b y cen t r i f u g a t i o n . T h e p e l l e t w a sh o m o g en i ze d i n Bu f f e r P (1 mM N aH C O 3 , p H 7 .6 , 0 .1 mM CaC1 2, 0 .2 mM A T P) an d d i a l y s edag a i n s t t h e s am e b u f f e r f o r 4 8 h . T h e s am p l e w as c l a r i f ied b y cen t r i f u g a t i o n a t 1 00 0 0 0 g fo r 2 h .T h e s u p e rn a t an t w as d i l u t ed t o 1 m g m1 -1 w i t h Bu f f e r P , mad e 0 .1 M i n KC1 an d 1 mM i n Mg CI~ ,a n d i n c u b a t e d a t r o o m t e m p e r a t u r e f o r 3 0 m i n t o a l l o w p o l y m e r i z a t i o n .

N - ( 1 - P y r e n y l ) i o d o a c e ta m i d e ( M o l e c u l a r P r o b e s , J u n c t i o n C i t y , O r e g o n , U . S . A . ) is s t o re dd e s i c c a t e d in th e d a r k a t - 1 0 C . 7 .5 m o l N - ( 1 - p y r e n y l ) i o d o a c e t a m i d e ( m o l. w t 3 8 5 ) w e r e a d d e dp e r m o l a c ti n . T h e N - ( 1 - p y r e n y l ) i o d o a c e t a m i d e w a s f ir s t d i s s o l v e d in d i m e t h y l f o r m a m i d e a t ac o n c e n t r a t io n o f 6 m g m 1- 1. A l t h o u g h t h e N - ( 1 - p y r e n y l ) i o d o a c e t a m i d e i m m e d i a t e l y p r e c ip i t a te sf r o m s o l u t i o n u p o n a d d i t i o n t o t h e a q u e o u s a c t i n s o l u t i o n , t h i s a p p r o a c h g a v e m o r e e f f i c i e n tl a b e ll i n g t h a n t h e a d d i t io n o f s o l i d N - ( 1 - p y r e n y l ) i o d o a c e t a m i d e . T h e m i x t u r e w a s r o t a t e d a t r o o mt e m p e r a t u r e i n t h e d a r k f o r 16 to 2 4 h.

T h e ac t i n f i l amen t s w e re co l l ec t ed b y cen t r i f u g a t i o n [ a s i zeab l e p e l l e t o f p r ec i p i t a t edN - ( 1 - p y r e n y l ) i o d o a c e t a m i d e a l s o s e d i m e n t s ] , t h e p e l l e t w a s h o m o g e n i z e d i n B u f f e r A , a n d t h em i x t u re d i a l y s ed ag a i n s t B u f f e r A fo r 4 8 h . A f t e r c la r i f ica t i o n (1 0 0 0 0 0 g x 2 h ) t h e u p p e r 3 / 4 o ft h e s u p e r n a t a n t w a s c h r o m a t o g r a p h e d o n S e p h a d e x G - 15 0 in B u f fe r A t o y i el d m o n o m e r i cp y r e n e - l a b e l l e d a c t i n.Extinction coefficient for py rene actinT h e p r o t e i n c o n c e n t r a ti o n o f p y r e n e a c ti n w a s m e a s u r e d u s i n g b o t h L o w r y et al. (1 9 5 1 ) an dBrad fo rd (1 9 7 6 ) p ro t e i n a s s ay s w i t h n a t i v e ac t i n a s t h e s t an d a rd . K o u y ama & Mi h as h i ( 1 : 9 8 1 )d e t e rm i n ed an ex t i n c t i o n co e f f i c i en t o f 2 2 0 0 0 M 1 cm - I a t 3 4 4 n m f o r t h e p y r e n e m o i e t y o fp y r en e ac t i n . F ro m o u r p ro t e i n d e t e rm i n a t i o n s a n d t h e ex t i n c t i o n co e ff i c ien t o f n a t i v e ac t i n a t2 9 0 n m ( 2 6 6 0 0 M 1 c m - 1 ; H o u k & U e , 1 9 7 4) w e d e t e r m i n e d t h e e x t in c t io n c o e f f ic i e nt o f p y r e n eac t in a t 2 9 0 n m t o b e 2 9 4 0 0 M 1 cm -1 . T h e re fo re , i n a mi x t u re o f p y ren e an d n a t i v e ac t i n o n e canc o r r e ct th e a b s o r b a n c e a t 2 90 n m b y s u b t r a c t i n g 0 . 1 2 7 t i m e s t h e a b s o r b a n c e a t 3 4 4 n m . T o t a la c ti n e q u a l s t h e q u o t i e n t o f c o r r e c te d a b s o r b a n c e a t 2 9 0 n m d i v i d e d b y 2 6 6 0 0 M 1 c m - 1 . O u rl a b e ll in g r a t io s w e r e r e g u l a r ly g r e a t e r t h a n 0 . 9 5 b u t n e v e r g r e a t e r t h a n 1 .0 0 .Fluorescence and light scatteringM e a s u r e m e n t s o f f lu o r e s c e n c e a n d l ig h t sc a t t er i n g w e r e m a d e i n a P e r k i n - E l m e r M o d e l 6 5 0 - 1 0 Ss p e c t r o f l u o r o m e t e r . T h e i n s t r u m e n t w a s e q u i p p e d w i t h a b r a s s , w a t e r - j a c k e t e d c u v e t t e h o l d e r

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P y r e n e a c ti n 2 5 5a n d a m a g n e t i c s t ir re r , d e s i g n e d a n d b u i lt w i t h t h e a d v i c e a n d a s s is t an c e o f D a n i e l K i eh a r t a n d E dH o r n . T h e c u v e t t e h o l d e r s u p p l i e d w i t h t h e i n s t r u m e n t w o u l d n o t a c c u r a t el y m a i n t a i n t h et e m p e r a t u r e o f th e s a m p l e . T h e t e m p e r a t u r e w a s 2 5 C , c o n t r o l l e d b y a c ir c u l at in g w a t e r b a t h .T h e ' n o r m a l ' m o d e , w h i c h c o r r e c ts fo r f l u c t u a ti o n s in la m p i n t e n s i ty , w a s e m p l o y e d . T o m i n i m i z ef l u o r e s c e n t b l e a c h i n g , a n e x c i t a t io n s i it w i d t h o f 3 n m w a s u s e d a n d a t r a n s l u c e n t g l a s s fi lt e r w a si n s e r te d b e t w e e n t h e l a m p a n d t h e s a m p l e : U n d e r t h e s e c o n d i t i o n s b l e a c h i n g o f a n a ct in p o l y m e rs o l u t io n w a s 1 % p e r h o u r , w h i c h a l l o w e d u s to m a k e c o n t i n u o u s m e a s u r e m e n t s . T h e e m i s s io n s li tw i d t h w a s 1 0 n m . T h e e x c i ta t io n a n d e m i s s io n w a v e l e n g t h s w e r e 3 6 5 a n d 4 0 7 n m , r e s p e c ti v e ly ,f o r f l u o r e sc e n c e m e a s u r e m e n t s a n d 4 5 0 a n d 4 5 0 n m f o r l ig h t sc a t te r i n g . T h e c u v e t t e w a s 1 0 m mb y 1 0 r a m , c o n s t r u c t e d o f g l as s. T h e s a m p l e s iz e w a s 1 - 1 . 5 m l . F o r fl u o r e s c en c e m e a s u r e m e n t sw e u s e d i m i d a z o l e G r a d e 111 ( S i g m a , S t L o u i s , M i s s o u r i , U . S . A . ) a s a p H b u f f e r .

Ostwald viscometryO s t w a l d c a p i ll a ry v i s c o m e t r y w a s p e r f o r m e d a s d e s c r i b e d p r e v i o u s l y ( C o o p e r & P o ll ar d , 1 9 8 2) .C a n n o n v i s c o m e t e r s n o . 1 5 0 ( S t at e C o l le g e , P e n n s y l v a n i a , U . S . A . ) i m m e r s e d i n a 2 5 C w a t e rb a t h w e r e u s e d . T h e s a m p l e s i z e w a s 0 .6 m l .

Elongation ra te constantsT h e e l o n g a t i o n r a te c o n s t a n t s w e r e m e a s u r e d i n a m a n n e r s im i la r to P o l la r d & M o o s e k e r ( 1 9 8 1 ).L o w c o n c e n t r a t i o n s o f m o n o m e r i c a c t i n f r o m 1 t o 7 / ~ M i n th e e x p e r i m e n t a l b u f f e r w e r e i n c u b a t e da t t i m e z e r o w i t h m o r p h o l o g i c a l l y i d e n t if i a b l e n u c l e i . F o r n a t iv e a c t i n t h e s e n u c l e i w e r eg l u t a r a l d e h y d e - f i x e d S l - d e c o r a t e d a c t in f i la m e n t ( d e v e l o p e d b y M a r s c h a l l S . R u n g e , d e s c r i b e d i nT s e n g & P o ll a rd , 1 9 82 ) a n d f o r p y r e n e a c t i n t h e y w e r e Limulus s p e r m a c r o s o m a l p r o c e s s e s ( T i ln e y e tal., 1 98 1) . T h e t w o t y p e s o f n u c l e i g i v e r e s u l t s s i m i l a r to e a c h o t h e r a n d s i m i l a r t o t h o s e f o r m i c r o v i l l a rc o r e s ( P o l la r d & M o o s e k e r , 1 98 1) . F o r e x a m p l e , w i t h Limulus p r o c e s s e s B o n d e r & M o o s e k e r ( 19 81 )f o u n d k + = 1 0 7 M 1 S 1 , k+P = 1 .5 x 1 0 6 M - 1 S ~ , k B = 1 . 5 s - 1 , a n d k_ = 0 . 5 s 1 f o r na t i v e a c t i n .T h e s e r e s u l t s a g r e e w i t h t h o s e r e p o r t e d h e r e f o r f i x e d S l - a c t i n f i la m e n t s , f o r m i c r o v i l l a r c o r e s( P o l l ar d & M o o s e k e r , 1 9 8 1 ) a n d , f r o m o u r l a b o r a t o r y , f o r f i x ed S l - a c t i n f i l a m e n t s ( R u n g e &P o l l a r d , u n p u b l i s h e d r e s u lt s ) . A t v a r i o u s t i m e s f r o m 2 0 to 4 8 0 s a f t e r t i m e z e r o , t h e r e a c ti o n w a ss t o p p e d b y t h e a d d i t i o n o f b u f f e r to d i lu t e t h e m o n o m e r i c a c t i n to a c o n c e n t r a t i o n n e a r i t s c r it ic a lc o n c e n t r a t i o n , a n d t h e d i l u t e d s a m p l e s w e r e q u i c k ly a p p l ie d t o a g l o w - d i s c h a r g e d , c a r b o n a n dP a r l o d i o n - c o a t e d g r id . T h e g r i d s w e r e n e g a t i v e l y s t a i n e d w i t h u r a n y l a c et a te o r u r a n y l f o r m a t e .E l e c tr o n m i c r o g r a p h s w e r e t a k e n , a n d t h e l e n g t h o f u n d e c o r a t e d a c t i n f i la m e n t g r o w t h a t e a c he n d o f a n u m b e r ( 1 0 - 6 0 ) o f n u c le i w a s m e a s u r e d . T h e l e n g t h w a s c o n v e r t e d t o ac ti n p r o t o m e r sa n d d i v i d e d b y th e r e a c ti o n ti m e to y i e l d r a te i n m o n o m e r s p e r s e c o n d . T h e m e a n a n d s t a n d a r dd e v i a t io n o f t h e r a te s a t e a c h o f f o u r ac t in m o n o m e r c o n c e n t r a t i o n s w e r e d e t e r m i n e d . T h e s ev a l u e s w e r e e m p l o y e d i n a l e a s t- s q u a r e s r e g r e s s io n t o a s in g l e li ne w i t h w e i g h t e d y v a l u e s to y i el dt h e s l o p e ( a s s o c i a t i o n r a te c o n s t a n t , k + ) a n d y i n t e r c e p t ( - k _ , n e g a t i v e d i s s o c i a t i o n r a te c o n s t a n t )a n d t h e s t a n d a r d d e v i a t io n f o r e a c h v a l u e.

Re su l t sCopolym erization at steady-state equilibriumA c o n v e n i e n t t e st f o r c o p o l y m e r i z a t i o n o f l a b e l l e d a n d n a t iv e a c ti n is t o m e a s u r e t h ec r it ic a l c o n c e n t r a t i o n o f a c t in m i x t u r e s w i t h v a r i e d r a t io s o f l a b e l l e d t o n a t i v e a c t in . I nt h e a b s e n c e o f c o p o l y m e r i z a t i o n , t h e cr it ic a l c o n c e n t r a t i o n , a s m e a s u r e d b yf l u o r e sc e n c e , w i l l d e p e n d o n l y o n th e p y r e n e a c ti n c o n c e n t r a t i o n a n d b e i n d e p e n d e n t o f

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F ig . 1 . C o p o l y m e r i z a t i o n o f n a ti v e a n d p y r e n e a c t i n . F l u o r e s c e n c e s i gn a l i n a r b i t ra r y u n i t s i sp l o t t e d v e r s u s t o t a l ac t in c o n c e n t r a t i o n . T h e p e r c e n t a g e o f p y r e n e l a b e l w a s 3 0 ( l l ) a n d 6 ( (9 ) . T h es i g n a l v a l u e f o r 6% w e r e m u l t i p l i e d b y f i v e b e f o r e t h e g r a p h w a s m a d e . T h e c r i ti c al c o n c e n t r a t i o ni s 0 .6 /~ M f o r e a c h s e t o f d a t a , w h i c h i m p l i e s t h a t c o p o l y m e r i z a t i o n o f p y r e n e a n d n a t i v e a c t i n h a so c c u r r e d . C o n d i t i o n s : 0 . 1 M K C I, 1 m M M g C 1 2, 0 . 2 m M A T P , 1 0 m M p o t a s s i u m p h o s p h a t e ,p H 7 . 0 , a n d 2 1 .5 % ( v / v ) B u f f e r A f r o m t h e a c t i n . T h i s e x p e r i m e n t w a s r e p e a t e d f o r t h r e e o t h e ri o n i c c o n d i t i o n s w i t h s i m i l a r a c t i n c o n c e n t r a t i o n s , a n d c o p o l y m e r i z a t i o n w a s f o u n d i n e a c h c a s e .T h o s e c o n d i t i o n s w e r e t h e f o l l o w i n g : (1 ) 0 .1 M K C 1, 1 m M M g C 12 , 2 0 m M i m i d a z o l e - H C 1 , p H 7 .0 ,an d 90% (v /v) B uffer A w i th ou t Ca2+; (2) 0 .1 M KC1, 1 mM MgC12, 20 mM im idazo le -H C1, pH7.0 , 90% (v /v) Buffer A; a nd (3) 0 .1 M KC1, 20 mM im idazo le-H C1, pH 7 .0, 90% (v /v) Buffer A.

n a t i v e a c t i n c o n c e n t r a t i o n . I f c o p o l y m e r i z a t i o n o c c u r s , t h e c r it ic a l c o n c e n t r a t i o n i n t e r m so f to t a l a c ti n w i l l b e t h e s a m e f o r al l r a t io s o f p y r e n e t o n a t i v e a c t i n. A r e p r e s e n t a t i v ee x p e r i m e n t i s s h o w n i n F ig . 1 . T h e c r it ic a l c o n c e n t r a t i o n s a r e th e s a m e f o r t w o m i x t u r e si n w h i c h t h e f r a c t i o n o f p y r e n e a c t i n d i f f e r s b y a f a c t o r o f 5. B y t h i s c r i t e r i o n ,c o p o l y m e r i z a t i o n a l so o c c u r s u n d e r t h e s e v e r a l o t h e r i o n i c c o n d i t i o n s g i v e n i n t h ec a p t i o n t o F i g . 1. T h e s l o p e s o f t h e l i n e s fo r t h e m o n o m e r a n d p o l y m e r s e c t i o n s o f t h ec u r v e s h o w t h a t th e f l u o r e s c e n t s i gn a l fr o m p o l y m e r w a s 7 - 1 0 - f o l d g r e a t er th a n t h es ig n al f r o m m o n o m e r .Elongation rate constantsW i t h i n t h e l i m i t s o f e x p e r i m e n t a l e r r o r t h e e l o n g a t i o n r a t e c o n s t a n t s f o r p y r e n e a c t in a ret h e s a m e a s f o r n a t i v e a c t i n ( T a b l e 1 ) .P olymerization kineticsT h e t i m e c o u r s e o f p o l y m e r i z a t i o n w a s m e a s u r e d u s i n g v a r i e d r a ti o s o f p y r e n e t o n a t i v ea c t in . I n o n e i o n i c c o n d i t i o n , w i t h M g 2+ p r e s e n t , t h e e a r l y s l o w p h a s e o f p o l y m e r i z a t i o n

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P y r e n e a c t i nT a b l e 1 . E l o n g a t io n r a t e c o n s t a n t s f o r p y r e n e a c ti n . E r r o r s h o w n i s o n es t a n d a r d d e v i a t i o n , c a l c u l a t e d a s i n M a t e r i a l s a n d m e t h o d s . C o n d i t i o n s :0 .1 M K C 1 , 2 0 m M i m i d a z o l e - H C l , p H 7 .0 , 2 5 % ( v / v ) B u f f e r A , 2 5 C .

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Na t i v e 9 .5 2 .4 4 .2 3 .7P o i n t e d e n d

P y r e n e 0 . 7 0 .3 - 0 . 1 0 . 5Na t i v e 1 .1 0 .2 0 .8 0 .3

i s r e l a t iv e l y fa s t a n d c u r v e s f o r p o l y m e r i z a t i o n o f 1 00 % , 1 0 % a n d 1 % p y r e n e a c t in w e r ea l w a y s s u p e r i m p o s a b l e , a s s h o w n in F i g. 2 a . I n t h e p r e s e n c e o f C a 2+ a n d t h e a b s e n c e o fM g 2+, t h e e a r l y s l o w p h a s e is r e l a ti v e ly s l o w a n d s u c h c u r v e s a r e ra r e l y s u p e r i m p o s e de x a ct ly . S o m e t i m e s t h e h i g h e r p e r c e n t a g e o f p y r e n e p o l y m e r i z e d f a s t e r b u t s o m e t i m e s i tw a s s l o w e r . W e s u s p e c t t h is d i f f e re n c e w a s d u e t o n u c l e a t i n g s p e c i e s w h i c h d e v e l o p e di n o u r p r e p a r a t i o n s o f p y r e n e a n d n a t i v e a c t i n s. F ig . 2 b is a r e p r e s e n t a t i v e e x p e r i m e n tw h i c h s h o w s t h a t t h e d i ff e r e n c e s b e t w e e n t h e c u r v e s a r e s m a l l. T e l l a m & F r i e d e n( 1 9 8 2) p e r f o r m e d o n e e x p e r i m e n t s im i la r t o t h o s e s h o w n h e r e. T h e y u s e d a n a r r o w e rr a n g e o f p y r e n e a c t in p e r c e n t a g e t h a n w e d id , a n d t h e y m a d e d i s c o n t i n u o u s i n s te a d o fc o n t i n u o u s m e a s u r e m e n t s .

/ / /O0 500 1000 1500Time (s)(a )

2000 2500

10

i ! 800 1~00'24'00 ' s~00'40 00Time (s)(b)F i g . 2 . P o l y m e r i z a t i o n k i n e t i c s o f v a r i e d r a t i o s o f p y r e n e t o n a t i v e a c t i n . F l u o r e s c e n c e s i g n a l inn o r m a l i z e d u n i t s i s p l o t t e d v e r s u s t i m e . ( a ) T h e c u r v e s r e p r e s e n t , f r o m l e ft t o r i g h t , 1 0 0 % , 1 0 %a n d 1 % p y r e n e a c ti n . T h e c u r v e s a r e d i s p la c e d 2 5 0 s e c o n d s f r o m e a c h o t h e r f o r c l a ri ty .C o n d i t i o n s : 7 .1 /~ M a c t in , 1 m M M g C 1 2 , 0 . 1 M K C 1 , 2 0 m M i m i d a z o l e - H C 1 , p H 7 . 0, 9 0% ( v / v )B u f f e r A . (b ) T h e c u r v e s r e p r e s e n t , f r o m l e f t t o r ig h t , 2 0 % , 1 0% , 1 % a n d 0 .2 % p y r e n e a c t i n . T h ec u r v e s a r e d i s p l a c e d 4 0 0 s e c o n d s f r o m e a c h o t h e r f o r c l a r it y . C o n d i t i o n s : 1 8 .9 /2 M a c t in , 0 . 1 MK C 1 , 2 0 m M i m i d a z o l e - H C I , p H 7 .0 , 9 0% ( v / v ) B u f f e r A .

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2 58 C O O P E R , W A L K E R a n d P O L L A R DC o n t i n u o u s a n d d i s c o n t in u o u s m e a s u r e m e n t o f p o l y m e r iz a t io n k i n et ic s w e r e

c o m p a r e d t o d e t e rm i n e w h e t h e r t h e m e a s u r e m e n t a f fe c ts p o l y m e r i z a t io n . T h e t im ec o u r s e o f p o l y m e r i z a t i o n i s t h e s a m e f o r c o n t i n u o u s m e a s u r e m e n t a n d f or d i s c o n t i n u o u sm e a s u r e m e n t w h e r e w e h a v e t h e s a m p l e i l lu m i n a t e d f o r 5 s a n d t h e n n o t il lu m i n a t e dfo r 25 s .Com parison of fluorescence and ligh t scatteringN i n e t y d e g r e e s l ig h t s ca t te r in g h a s b e e n s h o w n t o b e a u s e f u l a s s a y f or p o l y m e r w e i g h tc o n c e n t r a t io n u n d e r t h e s e c o n d i t io n s w h e r e t h e f il a m e n t s a re l o n g (W e g n e r & E n ge l,1 9 7 5) . W e m e a s u r e d t h e t i m e c o u r s e o f a c ti n p o l y m e r i z a t i o n b y f l u o re s c e n c e a n d b yl ig h t s c a tt e r in g . T h e r e s u l t s , s h o w n i n F ig . 3, a r e s u p e r i m p o s a b l e f o r t h e t w o a s s a y s .T h e c u r v e s h a v e t h e s i g m o i d a l s h a p e w h i c h w e h a v e a l w a y s o b s e r v e d f o r t h e k in e t ic s o fp o l y m e r i z a t i o n o f f l e s h g e l - f i lt e r e d a c ti n . T e l l a m & F r i e d e n ( 1 9 8 2 ) p e r f o r m e d a s i m i la re x p e r i m e n t c o m p a r i n g p y r e n e a c ti n f lu o r e s c e n c e t o r a p i d s e d i m e n t a t i o n .

~ 1 0

3 4

,--r 800 1600 2400 3200Time (s)

4 0 0 0

Fig. 3 . Co mp ar ison of polymer izat ion kinet ics assay ed by py rene actin f luorescence and l ightscattering. Light s ignal in norm al ized uni ts i s p lot ted on the ordinate v ersus t ime. Four smo othsigmoidal curv es, g enerate d by cont inu ous moni tor ing, are show n. The tw o on the left aref luorescence, and the tw o on the r ight are l ight scattering. The curves are super impo sable bu tare sepa rated by 200 secon ds for c lar ity .

Ostwa ld viscosi tyM e a s u r e m e n t s o f O s t w a l d c a p il la r y v is c o s i ty ar e a f u n c t i o n o f b o t h p o l y m e r l e n g t hd i s t r i b u t i o n ( C r a i g & P o w e l l , 1 9 8 0 ) a n d p o l y m e r w e i g h t c o n c e n t r a t i o n . T h e le n g t hd i s t r i b u t i o n i s p r o b a b l y a f u n c t i o n o f t h e r e l a ti v e ra t e s o f b r e a k i n g a n d a n n e a l i n g o ff il a m e n ts . T a b l e 2 s h o w s m e a s u r e m e n t s o f i nt ri n si c v i s c o s it y f or n a t iv e a n d p y r e n e a c ti nf i l a m e n t s . T h e r e s u l t s a r e i d e n t i c a l w i t h i n t h e l i m i t s o f e r r o r .Fluorescence and filamen t lengthT o d e t e r m i n e w h e t h e r f l u o r e s c e n c e i s s e n s i t i v e t o f i la m e n t l e n g t h d i s t r i b u t i o n t h ef l u o re s c e n c e o f a n u n s t i r r e d s a m p l e w a s c o m p a r e d t o th a t o f t h e s a m p l e w h e n it w a ss t ir r e d . A s t ir b a r i n t h e c u v e t t e s t i rr e d t h e s o l u t i o n . S t i rr i n g c r e a t e s s h e a r , w h i c h b r e a k s

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Pyrene actinTable 2. Intrinsic viscosity (cm3g 1) of pyrene and native actinpolymer. The values shown are the average from two experiments.Error shown is calculated by propagating error in time measurementsthrough the viscosity calculations. Intrinsic viscosity was calculated asthe slope of a plot of relative viscosity versus actin concentration at oneshear rate. Conditions: 0.1 M KC1, 20 mM imidazole-HC1, pH 7.0,90% (v/v) Buffer A, and magnesium chloride as shown in headings.Samples were incubated at 25 C overnight before measurements weremade.

Pyrene actin Native actinNo Mg2+ 880 -+ 30 890 + 301 mM Mg2+ 870 -+- 30 870 + 30

259

actin filaments; to show that stirring actually broke filaments we followed the kinetics ofpolymerizat ion versus time. Initially the sample was not stirred. At about 50% polymerformation the stirrer was tur ned on for 30 s. The polyme rization rate after stirring was 50times larger than the rate before stirring, whic h indicated that the filament num ber hadincreased by 50 times and therefore the average filament was broken into 50 pieces.Whe n a sample in which po lymeriza tion was complete was stirred, the value of thefluorescence did not change. Even though the filaments were 50 times shorter, thefluorescence was the same. Therefore, fluorescence was ind epe nde nt of filament lengt hdistribution in this range of length.

Di s cu s s i onIn 1981 Kouyama & Mihashi described the preparation of pyrene actin and pointed toits usefulness as a probe of actin polymerization and its high sensitivity. We feel it is ofthe utmost importance to document rigorously the validity of any assay for actinpolymerization in the light of previous difficulties in the design and interpretation ofsuch assays (Cooper & Pollard, 1982). This is particularly impor tan t for the pyrene actinfluorescence assay because the simplicity and straight forward interpretation of the assaymake it like ly that it will receive wide use.

We have performed several key experiments which establish pyrene actinfluorescence as a valid assay for polymerization. Pyrene actin is identical to native actinin its critical concentration, elongation rate constants, kinetics of polymerization andOstw ald viscosity. Experiments wit h different ratios of pyrene to native actin show thatthe two copolymerize both kinetically and at steady state. At steady state our resultsshow that two different mixtures of native actin and pyrene actin which differ byfive-fold in their pyrene percentage have the same critical concentration. This findingunequivocally demon strat es copolymerization. A critical concentration meas urem ent for0 or 100% pyren e actin is neithe r necessary nor sufficient to do cum ent co-polymerization. The signal for the mixture of lower pyrene actin concentration was

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2 6 0 C O O P E R , W A L K E R a n d P O L L A R Dm u l t i p l i e d b y 5 t o m a k e t h e g r a p h ( F ig . 1 ). T h e a g r e e m e n t b e t w e e n t h e t w o s e t s o fv a l u e s s h o w s t h a t t h e s i g n a l d o e s n o t d e p e n d o n i n t e r a c t i o n s f r o m o n e p y r e n e a c t i n t oa n o t h e r . T h a t i s, a p y r e n e a c t i n p r o t o m e r i n a f i la m e n t g i v e s th e s a m e s i g n a l w h e t h e r i ti s a d j a c e n t t o p y r e n e o r n a t i v e a c ti n . K i n e ti c a ll y , t h e a s s e m b l y o f a c ti n m o n o m e r s i n top o l y m e r i n v o l v e s s te p s o t h e r th a n e l o n g a ti o n . T h e s e s t e p s i n c l u d e n u c le a t io n a n dp o s s i b l y ac t iv a t io n a n d f r a g m e n t a t i o n ( P o l l a r d & C r a ig , 1 9 82 ). O u r r e s u l ts s h o w t h a tv a r i o u s m i x t u r e s o f p y r e n e a c ti n a n d n a t i v e a c ti n p o l y m e r i z e w i t h s i m i l ar k in e t ic s .

T h e c r it ic a l c o n c e n t r a t i o n , ~ , i s d e t e r m i n e d b y t h e r a ti o o f t h e e l o n g a t i o n r a tec o n s t a n t s , a s s h o w n in E q u a t io n 1 ( O o s a w a & A s a k u r a , 1 9 7 5) , w h e r e k+ i s t h e s u m o ft h e a s s o c i a t i o n ra t e c o n s t a n t s f o r t h e b a r b e d a n d p o i n t e d e n d s , a n d k _ i s a s i m il a r s u m o ft h e d i s s o c i a t i o n r a t e c o n s t a n t s

e ~ = k _ / k + (1)

I d e n t i t y o f t h e c r i ti c al c o n c e n t r a t i o n f o r n a t i v e a n d p y r e n e a c t in i m p l i e s t h a t t h e r a ti oo f t h e i r e l o n g a t i o n r a te c o n s t a n t s i s a l s o t h e s a m e , b u t r i g o r o u s c h a r a c t e r i z a ti o n o f t h em o d i f i e d a c t i n r e q u i r e s t h a t t h e e l o n g a t i o n r a t e c o n s t a n t s m u s t b e m e a s u r e d d i r e c t l y f o rs e v e r a l r e a s o n s . F i rs t, t h e i d e n t i t y o f t h e c r it ic a l c o n c e n t r a t i o n o n l y i m p l i e s t h e i d e n t i t yo f t h e r a t i o o f t h e e l o n g a t i o n r a t e c o n s t a n t s , a n d t h e i r a c t u a l v a l u e s m a y d i f fe r . S e c o n d ,t h e c r it ic a l c o n c e n t r a t i o n m e a s u r e m e n t i s a t s t e a d y s t a t e, r e f l e c ti n g a c o m b i n a t i o n o f t h ec ri ti ca l c o n c e n t r a t i o n a t t h e b a r b e d a n d p o i n t e d e n d s , w h i c h m a y n o t b e t h e s a m e i n t h ep r e s e n c e o f A T P h y d r o l y s i s ( W e g n e r , 1 97 6). T h i r d , s i n c e t h e c r it ic a l c o n c e n t r a t i o nm e a s u r e m e n t is m a d e a t s t e a d y s t a te i n t h e p r e s e n c e o f A T P h y d r o l y s is , t h em e a s u r e m e n t r e fl e ct s a n u n c e r t a in c o m b i n a t i o n o f in t e ra c t io n b e t w e e n a c t in s p e c i e sw i t h b o u n d A D P o r A T P . T h e r e f o r e , w e h a v e m e a s u r e d t h e e l o n g a t i o n r at e c o n s t a n t s o fp y r e n e a c t i n d i r e c t l y u s i n g a n e l e c t r o n m i c r o s c o p i c a s s a y ( P o l l a r d & M o o s e k e r , 1 9 8 1 )w i t h s h e a r e d a c r o so m a l p r o c e s s e s f ro m L i m u l u s s p e r m a s t h e m o r p h o l o g i c a l l yi d e n ti f ia b l e n uc l ei . T h i s a s s a y s e p a r a t e s b a r b e d a n d p o i n t e d e n d s a n d i s p e r f o r m e dr a p i d l y i n t h e p r e s e n c e o f e x c e s s A T P . T h e a b i l i ty o f a c r o s o m a l p r o c e s s e s t o n u c l e a t ep o l y m e r i z a t i o n w a s d e s c r i b e d b y T i l n e y e t a l . ( 1 9 8 1 ) . B o n d e r & M o o s e k e r ( t 9 8 1 ) a n do u r s e l v e s ( u n p u b l i s h e d r e s u l t s ) h a v e f o u n d t h a t t h e a c r o s o m a l p r o c e s s e s g i v e r e s u l t ss i m i l a r t o t h o s e u s i n g m i c r o v i l l a r c o r e s ( P o l l a r d & M o o s e k e r , 1 9 8 1 ) o r f i x e dS l - d e c o r a t e d a c t i n f i l a m e n t s ( u n p u b l i s h e d r e s u l ts , R u n g e & P o l la r d ) a s n u c l e i i nq u a n t i t a t i v e a s s a y s f o r e l o n g a t i o n r a te s . W i t h i n t h e l i m i ts o f e x p e r i m e n t a l e r r o r, t h ee l o n g a t i o n r a te c o n s t a n t s f o r p y r e n e a c ti n a r e th e s a m e a s f o r n a t i v e a c t in .T h e c o m p a r i s o n o f p y r e n e a c t i n f l u o r e s c e n c e w i t h l i g h t s c a tt e r in g c o n f i r m s t h a t t h ef l u o r e s c e n t s i g n a l i s p r o p o r t i o n a l t o p o l y m e r w e i g h t c o n c e n t r a t i o n . W e h a v e s h o w n ,f u r t h e r m o r e , t h a t t h e f l u o r e s c e n t s i g n a l f r o m a s t i rr e d s o l u t i o n o f a ct in f i l a m e n t s is t h es a m e a s t h a t f r o m a n u n s t i r r e d s o l u t i o n . T h i s c o n f i r m s t h a t t h e a s s a y i s n o t s e n s i t i v e t of il a m e n t l en g t h . W e h a v e c o n f i r m e d t h e h i g h s e n s it i vi t y o f t h e a s s ay . W e m e a s u r e d a ni n c r e a s e o f 7 - 1 0 - f o l d i n t h e f l u o r e s c e n c e b e t w e e n p o l y m e r a n d m o n o m e r , w i t he x c it a ti o n a t 3 6 5 n m a n d e m i s s io n a t 40 7 n m . K o u y a m a & M i h a s h i ( 19 8 1 ) e s t i m a t e d a

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P y r e n e a c t in 2 6 12 0 - 2 5 - f o l d f l u o r e sc e n c e i n c r ea s e f r o m t h e ir o b s e r v a t i o n t h a t t h e q u a n t u m y i e ld w i t he x c it a ti o n a t 3 42 n m i n c r e a s e d f i v e -f o ld . W e h a v e s h o w n t h a t w i t h a p p r o p r i a t ef il te r s b l e a c h i n g c a n b e m i n i m i z e d t o a l lo w c o n t i n u o u s m o n i t o r i n g o f t h e s i g n a l, as i gn i fi c an t i m p r o v e m e n t o v e r d i s c o n t i n u o u s m e a s u r e m e n t , w h i c h h a s t h e o r et i c al a n de x p e r i m e n t a l d i f fi c u lt i es . U n d e r t h e s a m e c o n d i t i o n s , t h e p o l y m e r i z a t i o n ra t e w a s n o ta f f e c t e d b y t h e e x c i t a t i o n l i g h t e m p l o y e d i n t h e f l u o r o m e t e r .

W e f e e l t h i s a s s a y i s s u p e r i o r t o o t h e r a v a i la b l e a s s a y s f o r s e v e r a l r e a s o n s . F i rs t, t h es i g n a l i s p r o p o r t i o n a l o n l y t o p o l y m e r w e i g h t c o n c e n t r a t i o n a n d n o t t o l e n g t hd i s t r i b u t io n , a d r a w b a c k o f c a p il l a r y v i s c o m e t r y . S e c o n d , t h e s a m p l e is n o t d i s t u r b e d ,a s i n f l o w b i r e f r i n g e n c e o r c a p i l l a ry v i s c o m e t r y . T h i r d , t h e s i g n a l is h i g h , b o t h r e l a ti v e tot h e m o n o m e r s i g n al a n d a b s o l u t e l y o n a s t a n d a r d f l u o r o m e t e r . T o c o n s i d e r o t h e r s im i l ara s s a y s , 7 - c h l o r o - 4 - n i t r o b e n z e n o - 2 - o x a - l , 3 - d i a z o l e a c t i n ( N B D - N E M - a c t in ) , a n o t h e rf l u o r e s c e n t d e r i v a t i v e , i n c r e a s e s i ts f l u o r e s c e n c e b y o n l y 2 . 2 -f o l d o n p o l y m e r i z a t i o n( D e t m e r s e t a l . , 1 98 1). R e s o n a n c e e n e r g y t r a n s f e r r e q u i r e s t h e s y n t h e s i s o f t w o f l u o r -e s c e n t a c t i n d e r i v a t i v e s , a n d i t s s e n s i t i v i t y h a s n o t b e e n c o m p a r e d t o o t h e r m e t h o d s( T a y l o r e t al . ,1981). A 23 2 m e a s u r e s a s m a l l c h a n g e ( 1 - 2% ) i n t h e t o ta l a b s o r b a n c e( H i g a s h i & O o s a w a , 1 96 5), w h i c h l i m i ts t h e q u a n t i t ie s o f o t h e r s u b s t a n c e s , s u c h a s p r o -t e in s a n d n u c l e o t i d e s , w h i c h c a n b e p r e s e n t i n t h e s a m p l e . T h e a s s a y a l so r e q u i r e s af i r s t- r a t e s p e c t r o p h o t o m e t e r . L i g h t s c a t t e r in g a l so h a s a h i g h s ig n a l , b u t t h e b a c k g r o u n d i so r d e r s o f m a g n i t u d e h i g h e r t h a n t h a t o f p y r e n e a c t i n f lu o r e s c e n c e, le a d i n g t o a lo w e rs i g n a l - t o - n o i s e r a ti o . L i g h t s c a t te r i n g s a m p l e s m u s t b e m e t i c u l o u s l y f re e o f d u s t , d e b r i sa n d b u b b l e s . L i g h t s c a t te r i ng a ls o m i g h t n o t b e p r o p o r t i o n a l t o p o l y m e r w e i g h tc o n c e n t r a t i o n i f t h e f i l a m e n t s a r e v e r y s h o r t , b u t p y r e n e a c t in f l u o r e s c e n c e s h o u l d b eu n a f f e c t e d . L i g h t s c a t t e r i n g i s a f f e c t e d b y f i l a m e n t a g g r e g a t i o n a n d f i l a m e n t b i n d i n gp r o t e i n s , s u c h a s t r o p o m y o s i n ( W a l s h & W e g n e r , 1 9 8 0 ).I n s u m m a r y , w e a r e p l e a s ed t o e x t e n d t h e w o r k o f K o u y a m a & M i h a s h i ( 1 98 1 ) t os h o w t h a t p y r e n e a c t i n i s a u s e f u l p r o b e f o r a c t i n p o l y m e r i z a t i o n . I t s s t r o n g p o i n t s a r et h e f o l l o w i n g : ( 1 ) t h e s i g n a l is p r o p o r t i o n a l t o p o l y m e r w e i g h t c o n c e n t r a t i o n o n l y ; (2 )t h e s a m p l e i s n o t s h e a r e d ; a n d ( 3 ) t h e s e n s i t i v i ty is h i g h . O n e p o t e n t i a l d r a w b a c k i s t h ee f f e c t o f t h e p y r e n e l a b e l o n t h e i n t e r a c t i o n o f a c t in w i t h o t h e r p r o t e i n s . K o u y a m a &M i h a s h i f o u n d t h a t h e a v y m e r o m y o s i n i n t e r a c t e d d i f f e re n t l y w i t h n a t i v e a s o p p o s e d t op y r e n e a c ti n . I n p r e l im i n a r y r e s u lt s w e h a v e f o u n d ( L ee e t a l . , 1 9 8 2 ) t h a t A c a n t h a m o e b ap r o f il i n b i n d s s i m i l ar l y t o p y r e n e a n d n a t i v e A c a n t h a m o e b a ac t in .

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