Adenosine 5′-phosphate and assembly of actin filaments in vitro

J. Mol. Biol. (1976) 107, 623-629

LETTER TO THE EDITOR

Adenosine 5'-phosphate and Assembly of Actin Filaments in Vitro Loewy (1952) reported that adding adenosine 5'-phosphate to crude supernatants of Physarum extracts caused small increments in their specific viscosity. It is shown here that 5'AMP t has this effect on the soluble fraction (100,000 g supernatant) and that the viscosity rises are correlated with the new appearance of individual filaments from amorphous precursor material. The filaments formed are shown to be actin by their size, shape and ability to interact with heavy meromyosin subfragment 1. ATP and inosine 5'-phosphate also cause filament assembly but 3'AMP is inactive. A polypeptide with a molecular weight of 55,000 is found in sodium dodeeyl sulfate/polyacrylamide gels of hybrid complexes made by co-precipitating supernatant proteins with added rabbit myosin. These hybrid complexes form 5'AMP when incubated with ATP. Two recent reports on very different cells show that actin can exist in cells in a non-filamentous state, even when conditions of protein concentration, ionic strength and temperature are such as to favor the polymerization of purified actin into filaments. Hinssen (1972) and Tilney et al. (1973) showed that in Physarum plasmodial sap and in the acrosomal process of certain echinoderm sperm, respectively, a precursor form of filamentous actin occurs. In both cases they observed a granular or amorphous material by electron microscopy. Tilney et al. (1973) found the precursor material to remain in solution after centrifugation at 80,000 g for over three hours. Hinssen (1972) demonstrated the presence of the actin precursor by conversion to paracrystalline arrays of actin filaments minutes after the addition of 50 mM-magnesium chloride. Tilney et al. (1973) showed rapid conversion in vivo of the acrosomal material into actin filaments in the acrosomal process ; however, the conversion would only go to a slight extent in vitro. To look for a possible physiological control of actin polymerization in cells, I re-investigated Loewy's (1952) early findings on the actomyosin-like properties of plasmodial extracts. He reported a small but distinct increase in viscosity of crude supernatants of extracts of Physarum kept at room temperature to which A T P was added (a slow viscosity rise followed an initial very rapid drop in viscosity) or to which 5'AMP t was added; in which case no initial drop was observed. I confirmed his results with the soluble fraction from Physarum and found t h a t the viscosity increments could be correlated with the new appearance in the extracts of m a n y flee filaments which have been identified as actin. Precipitation of supernatants with muscle myosin shows that a polypeptide with a molecular weight of 55,000 co-precipitates with the hybrid complex of actin and myosin. These results are reported at this early stage because of current interest in actin polymerization in high sucrose - A T P extracts of non-muscle cells (Kane, 1975; Pollard, 1976; Stossel & Hartwig, 1976). Migrating Physarum plasmodia were used. Plasmodia were homogenized with a motor-driven Teflon pestle for three minutes with an equal volume of 1 M-KC1, t Abbreviations used: 5'AMP, adenosine 5'-phosphate; 3'AMP, adenosine 3'-phosphate. 623

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0"1 •-imidazole, 32 mM-ethyleneglycol-bis (~-aminoethyl ether) N,N'-tetraacetic acid (pH 7.6). The homogenates were centrifuged at 100,000 g (average) for 100 minutes. Lipid was removed and the clear yellow supernatant (5 to 6 mg protein]ml) used on the same day. Viscosity measurements were made hi a bath kept constant at 26~ • 0.02 deg.C, in 0.9 to 1-ml precalibrated viscometers with outflow times of 30 to 50 seconds. For electron microscopy, samples were rapidly diluted to the optimal concentrations for negative staining. Negative contrast was with 1 ~ (w/v) uranyl acetate (Huxley, 1963). Myosin or the heavy meromyosin enzymatic subfragment 1 were prepared b y the methods of Huxley (1963) and Margossian & Lowey (1973), respectively. Grids were examh~ed in an Hitachi HS-7S electron microscope equipped with a 50 /xm gold foil objective aperture at an initial magnification of 20,000 • Comparisons were made on identically diluted samples, and dilutions were performed directly before grids were prepared. The adenosine 5'-phosphate, adenosine 3'-phosphate, inosine 5'-phosphate and ATP were reagent chemicals from Sigma Chemical Company. Protein concentrations were estimated by the micro method of Lowry et al. (1951). Samples in sodium dodecyl sulfate were electrophoresed on gels as described by Laemmli (1970). Thin-layer chromatography was carried out on Eastman 13254 20-cm strips, and ATP or ADP separated well from 5'AMP. Figure 1 shows the results of two representative viscometry experiments. As observed by Loewy (1952), 5'AMP caused viscosity increments in extracts from fresh or frozen plasmodia followed b y rapid drops after A T P addition. In the first experiment grids were taken from frozen mold extract at the beginning and end of the experiment as shown in Figure 1. The supernatant as viewed by electron microscopy consisted of amorphous aggregates and granular material. A typical view is shown in Figure 2(a). Short filaments were seen after incubations of 10 or 15 minutes with 5'AMP. The filaments shown in Figure 2(b) are from an experiment in which the supernatant was treated with 5'AMP for two hours, stored overnight at 5~ and the filaments concentrated by centrifugation at 80,000 g. Centrifugation of control supernatant stored overnight brought down only amorphous material (Fig. 2(c)). In Figure 2(d), the filaments are shown as they appeared in the supernatant 3000 seconds after adding 5'AMP to 10 mM final concentration. The insert in Figure 2(e) shows a single untreated filament on the left and on the right a filament from a grid treated with subfragment 1. Although sharp images were not obtained from this crude extract, arrowhead structures are nevertheless visible. Filament assembly did not occur if the extract was kept on ice. A few short filaments, variable in number, formed in some control extracts kept at room temperature but no significant rise in viscosity was observed. Average filament lengths increased with time: in one experiment from a mean of 272 nm at 103 seconds (N = 23) to 574 nm at 3 • 103 seconds (N = 14). A T P also induced filament formation after the viscosity rise, but filaments did not disappear during the rapid viscosity drop after addition of ATP as shown in Figure 1, and counts showed that no measurable loss of filaments occurred. I t is likely then, t h a t the drop represents the well-known dissociation of myosin from actin filaments ; it is to be expected that myosin is present in this high-speed high-salt supernatant. Adenosine 3'-phosphate did not cause

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Time (s) FIG. 1. Changes in the specific viscosity of the 100,000 g supernatant on addition of 5'AMP, ATP or 3'AMP. ( 0 ) Data from an experiment using frozen m y x o m y e e t e ; (O, A) data from an experiment using fresh myxomycete. Note the arrows marking additions of nueleotides to the final concentrations stated. Arrows marked grid8 indicate the points at which samples were taken for electron microscopy. As noted in the text, in the frozen-mold experiment samples were taken at the beginning and end of the experiment. I n the fresh-mold experiment, samples were taken a t the start, at the point of maximLum rise and after the ATP drop. Grids taken at 3000 s in this experiment gave results shown in Fig. 2(d) ; in a similar control experiment the results are shown n Fig. 2(a).

filament assembly and as shown in Figure 1 we confirmed Loewy's (1972) finding t h a t it did not cause viscosity increments. Cytochalasin B (10 mg/ml) did not prevent the viscosity rise or the filament assembly due to 5'AMP. As both 5'AMP and ATP are effective in filament assembly," it is likely t h a t either one of them is the essential factor, or that both are acting through a third factor, perhaps ADP. Several experiments suggested but do not conclusively prove that ADP production was not necessary for filament formation. I f the reaction was carried out with 10 m•-sodium fluoride, there was no effect on the rate of viscosity rise, the final level, or the number of filaments per arbitrary field produced by adding 5'AMP. The rate of viscosity rise with inosine 5'-phosphate occurred at almost the same rate as with 5'AMP without any time lag, and the number of filaments produced was similar. Dialysis of the supernatant overnight against 100 volumes of 0.5 M-KCI, 0.05 M-imidazole (pH 7.6), preserved the effect of 5'AMP. These variations would act to decrease concentration of substrates, inhibit or side-step possible enzymes such as aldolase or enolase which could be activated b y 5'A_MYPto cause production of ADP or A T P from endogenous sources. ADP was also much less effective than 5'AMP in producing viscosity increments. However, since the viscosity effect is complex, depending on length of filaments and myosin-association, if ADP produced

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Fro. 2. All p r i n t s are of n e g a t i v e l y s t a i n e d p r e p a r a t i o n s f r o m Physarum e x t r a c t s d i l u t e d 1 : 1 1 w i t h t h e a p p r o p r i a t e buffer. (a) T h e 100,000 g supernatarLt a f t e r i n c u b a t i o n a t 25~ for 3000 s w i t h no a d d i t i o n s , m a g n i f i c a t i o n 60,000 • (b) A n o t h e r s a m p l e of s u p e r n a t a n t , t r e a t e d w i t h 10 mM-5"AMP for 3000 s a t 26~ dialyzed a g a i n s t 0-05 M-KCI, 0.005 ~[-imidazole for 18 h a t 5~ a n d c e n t r i f u g e d a t 80,000 g for 1 h. T h e t o p of t h e pellet, m a g n i f i c a t i o n 42,000 • (c) S a m e f r a c t i o n as in (b) b u t w i t h a p h o s p h a t e buffer t r e a t m e n t in place of 5 ' A M P . O t h e r w i s e identical t r e a t m e n t , m a g n i f i c a t i o n 100,000 x . (d) T h e 100,000 g s u p e r n a t a n t a f t e r s t a n d i n g a t r o o m t e m p e r a t u r e for 3000 s w i t h 10 m~[-5'AMP. C o m p a r e w i t h (a), m a g n i f i c a t i o n 70,000 • (e) T h e i n s e r t shows, on t h e left., a f i l a m e n t f r o m a n e x p e r i m e n t as in (d) a n d on t h e r i g h t , a n identical p r e p a r a t i o n t r e a t e d on t h e grid w i t h h e a v y m e r o m y o s i n s u b f r a g m e n t 1, m a g n i f i c a t i o n 70,000 •

LETTER

TO T H E E D I T O R

627

some A T P , t h e v i s c o s i t y m i g h t be lowered. L o e w y (1952) s h o w e d t h a t t h e v i s c o s i t y rise w i t h A T P followed t h e a p p e a r a n c e of n e a r l y t w o p h o s p h a t e s p e r m o l e o f A T P . This finding t o g e t h e r w i t h r e c e n t studies of K a w a m u r a & N a g a n o (1975) shows t h a t a s t r o n g a p y r a s e a c t i v i t y occurs in t h e soluble fraction. W e h a v e f o u n d t h a t w h e n A T P is i n c u b a t e d w i t h t h e soluble f r a c t i o n a n d c h r o m a t o g r a p h e d a s p o t w i t h t h e RF v a l u e o f a u t h e n t i c 5 ' A ~ P a p p e a r s as A T P d i s a p p e a r s ; b u t w h e n A M P is used, no spots in t h e p o s i t i o n o f A T P a n d A D P a p p e a r . T h e filaments f o r m e d were r e l a t i v e l y stable, since t h e y could be collected h,y f u r t h e r c e n t r i f u g a t i o n a t 80,000 g. Therefore i t seems u n l i k e l y t h a t t h e y were i n i t i a l l y b r o k e n d o w n into f r a g m e n t s b y t h e p r e p a r a t i v e u l t r a c e n t r i f u g a t i o n . To see if a n o t h e r

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FIG. 3. Major polypeptides stained by Coomassie brilliant blue after electrophoresis in 10% (w/v) acrylamide gels in the presence of 0.1% (w/v) sodium dodecyl sulfate. (1) Pellet fl'om (b) of Fig. 2; (2) 5 ~g each of muscle myosin, actin and carbonic anhydrase standards; (4) 5 ~g aetin standard alone; (3) and (5) 2 concentrations of a precipitate made by adding myosin to the supernatant as in (d) of Fig. 2, followed by dialysis to low ionic strength buffer and centrifugation at 10,000 g for 15 min. Molecular weights ( • 10-3) are indicated.

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protein was associated with the actin, the supernatants were dialyzed against low ionic strength buffer after addition of muscle myosin. After 18 hours the "hybrid" precipitates were collected at low speeds, rinsed and examined on 10% sodium dodecyl sulfate/polyacrylamide gels after electrophoresis. In addition to the major band in the position of actin, a second band at a molecular weight of 55,000 was present (Fig. 3). This was very consistent, but its intensity varied and in some runs (as here) lower molecular weight material is present. Note t h a t it is not present in the actin filaments pelleted after 5'AMP formation. The 55,000 molecular weight band was significantly decreased in some experiments when the hybrid was made after filament formation had gone to completion. Purified _Physarum actin polymerizes on the addition of salt in the presence of ATP, splitting the ATP to ADP and inorganic phosphate (Hatano et al., 1967; Adelman & Taylor, 1969) as does muscle actin. The actin present in the high-salt high-speed supernatant seems to require 5'AMP for polymerization although it is not certain that the nucleotide has a direct effect. A workhlg hypothesis to explain this curious reqlfirement is t h a t the 5'AMP does not act directly on actin but t h a t it releases a block to actin polymerization, perhaps by interaction with a factor which itself interacts with actin or with actomyosin. There are two known possible proteins which are in a position to affect actin or actomyosin. One is the actinin described by Hatano & Owaribe (1976). They find t h a t this protein combines with pure actin to form a low viscosity polymer. As the actinin has the same molecular weight on sodium dodecyl sulfate/polyacrylamide gels as aetin we would not be able to distinguish it in our experiments. A second candidate is the 55,000 molecular weight material found in our gels. A recently characterized calciumactivated ATP phosphohydrolase (Kawamura & Nagano, 1975) has this subunit weight and converts ATP into 5'AMP and pyrophosphate. I t is present in the soluble fraction of Physarum. We therefore tested the ability of the hybrids to form 5'AMP from ATP and found that a spot of the same RF value as authentic 5'AMP was formed by hybrids made from starting supernatants. I t is very likely therefore, that this band does represent the phosphohydrolase. In summary, Loewy's (1952) original finding represents an assembly process of actin, induced by 5'AMP (or inosine 5'-phosphate) upon a precursor complex whose composition is not yet clear. An enzyme capable of forming 5'AMP from A T P coprecipitates with hybrid actomyosin and is therefore suspected of playing a role in the assembly. This work was supported by grant no. AM-17492 and in part by grant no. HL-15835 from the National Institutes of Health. I am indebted to Ms A. Lombardo for excellent assistance. I thank Drs L. G. Tilney, A. G. Loewy, D. Kessler, S. Hatano and M. R. Adelman for stimulating and helpful discussions. Department of Anatomy, School of Medicine 9 University of Pennsylvania, Philadelphia, Penn. 19174, U.S.A.

VIVIAmVE T. NACH~AS

Received 24 February 1976 REFERENCES Adelma~, M. R. & Taylor, E. W. (1969). Biochemistry, 8, 4976-4988. Hatano, S., Totsuka, T. & Oosawa, F. (1967). Biochim. Biophys. Aeta, 146, 109-127. Hatano, S. & Owaribe, K. (1976). Cold Spring Harbor Conference on Cell Proliferation, vol. 3, pp. 449-511, Cold Spring Harbor Laboratory, New York.

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Hinssen, H. (1972). Cytobiologie, 5, 146-164. H u x l e y , H. E. (1963). J . Mol. Biol. 7, 281-308. K a n e , R. E. (1975). J . Cell Biol. 66, 305-315. K a w a m u r a , lVl. & Nagano, K. (1975). Bioehim. Biophys. Acta, 397, 207-219. Laemmli, II. K . (1970). Nature (London), 227, 680-685. Loewy, A. G. (1952). J . Cell Comp. Physiol. 40, 127-156. Lowry, O. It., Rosebrough, N. J., F a r r , A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265-275. Margossian, S. A. & Lowey, S. (1973). J . Mol. Biol. 74, 313-330. Pollard, T. D. (1976). J. Cell Biol. 68, 579-601. Stossel, T. D. & Hartwig, J. H. (1976). J. Cell Biol. 68, 602-619. Tilney, L. G., H a t a n o , S., Ishikawa, H. & Mooseker, M. S. (1973). J. Cell Biol. 59, 109-126. Note added in proof. Some of these findings were presented a t the 1st I n t e r n a t i o n a l Congress on Cell Biology held in Boston, September 1976 (J. Cell Biol. 79, 249a).