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Bundling of myosin subfragment-1-decorated actin filaments
J. Mol. Bid. (1987) 195, 351-358
Bundling
of Myosin Subfragment-l-decorated Actin Filaments Toshio Andot Cardiovascular Research Institute University of California San Francisco, CA 94143, IJ.S.A.
(Received 29 July 1986, and in revised form
2 January
1987)
We have reported previously that rabbit skeletal myosin subfragment-l (S-l) assembles actin filaments into bundles. The rate of this reaction can be estimated roughly from the initial rate (V,) of the accompanying turbidity increase (“super-opalescence”) of the acto-S-l solution. V, is a function of the molar ratio (r) of S-l to actin, with a peak at r = l/S to l/7 and minimum around r = 1. In the present paper we report a different type of opalescence (we call it “hyperopalescence”) of a&o-S-l solutions, which also resulted from bundle formation. Adjacent filaments in the bundles had a distance of approximately 180 8. Hyper-opalescence occurred at r x 1 when KCOOCH, was used instead of KCl. By comparing the effects of ADP, .+ADP, tropomyosin or ionic strength upon the super- and hyper-opalescence, we concluded that the two types of S-l-induced actin bundling had different molecular mechanisms. The hyper-opalescence type of bundling seemedto be induced by S-l, which was not complexed with actin in the manner of conventional rigor binding. The presence of the regulatory light chain did not affect hyper-opalescence (or super-opalescence), since there were no significant differences between papain S-l and chymotryptic S-l with respect to these phenomena.
is about 90 A. Initially, they observed that, when an appropriate amount of S-l was added to F-actin Actin is ubiquitous in eukaryotic cells. In nonsolutions, an instant enhancement of lightmuscle cells, actin exists in a variety of forms (i.e. scattering (due to S-l binding to F-actin) was monomer, filament, bundle, and mesh-work strucfollowed by a gradual increase of the scattering ture) and can change its form when required. with time. This gradual increase was called “superAssembly of actin filaments into higher-order opalescence”. The initial rate of super-opalescence structures is assisted by a variety of actin-binding was not proportional to the amount of S-l added, proteins such as fascin, fimbrin, villin, filamin, and but had a peak at an S-l to actin molar ratio (r) of a protein identified in lung macrophages (for l/S to l/7. The rate was nearly zero at r values reviews, see Weeds, 1982; Korn, 1982; Sheterline, above 0.5. In the course of the experiments, it’ was 1983). Heavy meromyosin prepared from skeletal noticed that the rate of super-opalescence was muscle myosin cross-links actin filaments in vitro by enhanced drastically, simply by replacing KC1 with binding each S-l portion to a different actin KCOOCH3. Not only was the magnitude changed, filament to form a raft-like structure (Trinick & but so also was the behavior of the rate as a Offer, 1979). It has been found by Ando & Scales function of r. Once r approached 1, the light-scattering (1985) that even the single-headed subfragment of intensity of the acto-S-l solutions increased rabbit skeletal myosin, S-1$, can assemble actin abruptly. Since this behavior is so peculiar, we can filaments into bundles whose interfilament distance expect that some unknown interactions between the proteins must be involved. In order to investigate t Present. address: Department of Physics, Faculty of such interactions, we have characterized acto-S-l Science, Kanazawa University, 1-l Marunouchi, solutions that contain acetate as a predominant Kanazawa, Ishikawa 920 Japan. anion, by studying the effects of nucleotides, $ Abbreviations used: myosin S-l, subfragment- 1; tropomyosin (TM), and ionic strength on the lightTM. tropomyosin; TES, N-tris[hydroxymethyl]methylscattering behavior, and by observing acto-S-l with 2-aminoethane sulfonic acid; E-ADP, 1 ,N6an electron microscope. ethenoadenosine 5’-diphosphate. 351 1. Introduction
OO22-2836/87/100351-08
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1987
Academic
Press
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(London)
Ltd.
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T. Ando according to the methods of Bailey (1948) and Ebashi et al. (1971), respectively. The molar concentrations of papain S-l, chymotryptic S-l and F-actin were determined by their ultraviolet absorption in 0.6 M-NaCl using E&& = 8.3, Ei& = 7.5 and Ei& = 65, respectively. and respective molecular weights of 1.33 x 105, 1.15 x lo5 and 4.2 x 104. Corrections for turbidity were made as described (Ando, 1984). The molar concentrations of TM and troponin were determined by the method of Lowry et al. (1951), taking values of 7.0 x lo4 and 7.5 x IO4 for their respective molecular weights. (b) Light-scattering
20
40
60
I 80
I
KCI, KEr. KNO, hd
Figure 1. Effects of KCl, KBr and KNO, upon the initial rate (V,) of super-opalescence. F-actin (3 PM) and chymotryptic S-l (0.45pM) were mixed in a solution containing 20 mM-TES (pH 7.0), 1 ITIM-UU3gneSiUm acetate, 0.2 m&r-calcium acetate, 0.1 mxl-dithiothreitol, 0.1 mM-KaKs and various amounts of one of the neutral salts to be tested (0, KCl; A, KBr; 0, KNO,) and potassium acetate at 20°C. The sum of potassium acetate concentration and the neutral salts concentration was kept at 0.1 M.
As described by Ando & Scales (1985), the lightscattering intensity of acto-S-l solutions was measured at 400 nm using a Hitachi Perkin-Elmer MPF4 fluorometer. Since the units of light-scattering intensity are arbitrary, the intensity was normalized by assigning a value of 10 to the intensity of 5 p&r-F-actin in a solution containing 0.1 M-KCl, 20 miw-TES (pH 7.0), 2 mM0.2 miw-Ca(CGGCH,),, 0.2 mm-dithioMg(COOCH,),, threitol and 0.1 mM-NaN, at 15°C. A small volume (5 to 160 ~1) of S-l solution was added to 2 ml of F-actin solution in a thermostatted fluorescence cuvette. The solution was mixed by sucking and blowing several times with a l-ml Pipetman automatic pipet, and the timecourse of changes in light-scattering intensity was immediately recorded on time-scanning chart paper. The initial rate (V,) of super-opalescence or hyper-opalescence was read from the chart paper. (c) Electron microscopy
2. Materials and Methods (a) Protein preparations Myosin was prepared from rabbit skeletal back and leg muscles (Tonomura et al., 1966) and stored in 0.3 M-KCl, 50% (v/v) glycerol at -20°C. Papain S-l was obtained by digesting myosin with insoluble papain in a solution containing 0.1 M-ammonium acetate (pH 7.2), 2 mMMgCl,, and 0.2 miw-dithiothreitol, and purified by the use of DEAE-Sepharose CL-6B. The purified S-l was concentrated using Sephadex G-25 powder. Chymotryptic S-l was obtained by the method of Weeds & Taylor (1975). S-l(A1) and Sl(A2) were separated using preparative DEAE-cellulose high-pressure liquid chromatography (h.p.1.c.). The eluted S-l was concentrated by 60% saturation of ammonium sulfate. After removal of the ammonium sulfate, the concentrated S-l was lyophilized in 0.15 M-SUCrOSe, 50 tIIMn-a~IUOniUIn acetate and 05 mw-dithiothreitol at pH 7.0. The lyophilized S-l was stored at -20°C. Just before use, the lyophilized S-l was dissolved with a small volume of the desired buffer solution, and clarified by centrifuging for 90 min at 47,000 revs/min using a 50 rotor. The h.p.1.c. and lyophilization steps were omitted in some cases. However, this omission did not significantly affect the results given below. Acetone powder of rabbit skeletal muscle was prepared as described (Ando & Asai, 1979). Actin was first purified by the method of Spudich & Watt (1971), and further purified by Sephacryl S-300 gel chromatography. A high concentration (15 mg/ml) of F-actin was stored in 0.1 mmATP, 20 mM-KCOOCH,, 20 miw-TES (pH 7.0). 2 mM-Mg(COOCH,),, 0.2 mMCa(COOCH,),, 0.2 m&r-dithiothreitol, 0.1 mmNaru’, at 6°C. Just before use, the stored F-actin was diluted to approx. 20 pM with an ATP-free buffer solution. TM and troponin were prepared from rabbit skeletal muscle
Droplets of solutions of 1 mw-F-actin plus 1.5 PM-S-~ were applied to freshly carbon-coated Formvar grids. After 30 s, a few drops of 1 ye (w/v) uranyl acetate were applied to the grid for another 30 s. The negative stain was then removed with torn filter paper. The grids were allowed to dry for several minutes before they were observed at 80 kV in a Philips EM 200 electron microscope.
3. Results (a) Further
characterization
of super-opalescence
When super-opalescence of acto-S-l solutions was first studied (Ando & Scales, 1985), KC1 was used for setting an appropriate ionic strength. Here, however, Cl- was found to decelerate super-opalescence (see Fig. 1). F-actin (3 ,UM) and chymotryptic S-l (0.45 PM) (i.e. r = 0.15) were used in this experiment. The sum of KCi and KCOOCH, concentrations was kept at 0.1 M. V, with 0.1 M-KCOOCH, was about 15-fold higher than that with 0.1 M-KCl. Therefore, unless otherwise mentioned, KCOOCH3 was used instead of KCl. The inhibitory effect of and NO;) was also tested. As other anions (Brshown in Figure 1, these anions inhibited superopalescence with the order of increasing effectiveness being Cl- < Br- < NO;. V, decreased when ADP had been preincubated in F-a&in solutions before adding chymotryptic S-l. V, decreased to zero with increasing ADP concentration (Fig. 2). V,, decreased by half at approximately 20 PM-ADP. This value is not very
Acto-S-l
353
Bundling
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0.2
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0.4 ADP, E -ADP
0.6
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Figure 2. Effect of preincubation of F-actin with ADP or E-ADP on the initial rate of super-opalescence. Chymotryptic S-l (0.45 PM) was added to 3 pni-F-actin amounts of ADP/e-ADP, preincubated in various 60 mM-potassium acetate, 20 mM-TES (PH 74L acetate, 0.2 mM-calcium acetate, 2 mM-magnesium 0.1 mw-dithiothreitol, and 0.1 mM-NaNu’,, at 20°C. 0. ADP effect; 0, E-ADP effect.
different from the reported values of the dissociation constant of Mg 2+ -ADP for rabbit skeletal actoS-l: 37 PM (Highsmith, 1976), 143~~ (Greene & Eisenberg, 1980). When E-ADP was used instead of ADP,
V, decreased
by
half
at
approximately
270 PM-E-ADP. This value is also not far from the dissociation constant of Mg’+-&-ADP (350 /AM) for rabbit skeletal acto-myosin in fibers reported by Yanagida (1981). Therefore, the acto-S-l-bound nucleotides seem to be responsible for the reduction of V,. This conclusion is plausible, considering that V, is not, a linear function of the amount of nucleotide-free acto-S-l complex. On the other hand, when ADP was added to a&o-S-l solutions that had been preincubated and were already showing super-opalescence, the lightscattering intensity stayed at a level slightly lower than just before ADP was added (Fig. 3). Since mechanical perturbation alone of acto-S-l solutions by mixing with a Pipetman reduced the superopalescencelevel, the slight reduction observed here was not due to ADP addition. Therefore, we can conclude that ADP addition to actin bundles cannot disassemble the bundles, although ADP inhibits bundle formation. TM had similar effects on the super-opalescence of acto-S-l solutions. TM addition to actin bundles cannot disassemble the bundles whereas TM inhibits bundle formation. A molar ratio of TM/a&in of as little as approximately 0.018 was sufficient to reduce V, by half (Fig. 4). Troponin had little influence on the TM effects on super-
0
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I 4
I 6
I 8
I IO
I I2
I 14
Time (mini
Figure 3. Effect on the super-opalescence of ADP addition to the acto-S-l solution that has shown superopalescence. At arrow 1, 0.45 PM-chymotryptir S-l was added to 3 PM-F-actin in a solution containing 0.1 M-potassium acetate, 20 mM-TES (pH 7.0), 1 mMmagnesium acetate, 0.2 mM-calcium acetate, 0.1 mMdithiothreitol, 0.1 mM-NaN,, at 20°C. At arrow 2. 0.2 mM-ADP was added.
opalescence, irrespective of the presence of Ca’+. To study the effect of the regulatory light chain on super-opalescence, the previous experiments were repeated using papain-S-1 instead of chymotryptic S-l. It gave essentially the same results as those obtained with chymotryptic S-I. (b) Hyper-opalescence We have reported (Ando & Scales, 1985) that V, reaches a peak at r = l/S to l/7, and then steadily decreasesto nearly zero when r is further increased (see Figs 2 and 3 of Ando & Scales, 1985). Though V, data at r above 0.7 were not shown in that paper, V, was zero at this range of r values (Fig. 3 of Ando & Scales (1985) implies this fact). Now, V, versus r was re-studied in solutions that did not contain Cl-, but contained CH,COO- instead. As shown in Figure 5 (chymotryptic S-l was used), V, reached a peak at r = l/6 to l/7 and then decreased to zero when r was increased (the first phase). However, as r was further increased, V, st#arted increasing very rapidly just’ before r reached the value 1.0, and reached a plateau at r z 2.5 (the second phase). The plateau value of V,, at r z 2.5 was about 14 times larger than the peak value of the first phase. The contrast between the present and previous observations of V, behavior around r = 1.0 resulted only from the difference of anion (i.e. Cl- or CH,COO-). Henceforth, in order to
354
T. Ando
I
I
I
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0.08
0.04
0
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0.12
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TM Q.LM)
Figure 4. Effect of preincubation of F-actin with TM on the initial rate of super-opalescence. Chymotryptic S-l (0.45 PM) was added to 3 PM-F-a&in plus various amounts of TM in a solution containing 60 mm-potassium acetate, 20 mM-TES (pH 7.0), 2 m&r-magnesium acetate, 0.2 mm-calcium acetate, 0.1 m&r-dithiothreitol, O-1 mMNaN,, at 20°C. distinguish the second phase from the first phase, we call the second one “hyper-opalescence” and we use “super-opalescence” for the first one. (c) Bundle
formation
Does hyper-opalescence also result from formation of bundles by S-l-decorated F-actin? In order to study this question acto-S-l was observed with an electron microscope. An acto-S-l sample (1.5 PMchymotryptic S-l plus 1 pi%-F-actin) was incubated at room temperature for one hour in a solution containing 0.1 M-KCOOCH,, 20 mM-TES (pH 7.0), 1 miw-Mg(COOCH,),, 0.2 miw-Ca(COOCH,),, 0.2 m&r-dithiothreitol, O-1 mM-NaN,, and then mounted on grids. As shown in Figure 6(a), wellordered bundles containing a few S-l-decorated actin filaments were observed. The bundles possessed distinct transverse stripes with a periodicity of about 350 A and an interfilament distance of about 180 A. It was difficult to see any arrowhead polarity of an S-l-decorated filament within the bundles. Fortunately, we had a few specimens of bundles in which two adjacent filaments branched at an end, so that we could clearly see the polarity (Fig. 6(b), (c), (d)). The arrowheads of both branched filaments pointed in the same direction (i.e. to the junction ((b), (c)), or to the splay direction (d)). That is, actin filaments within these particular specimens had parallel polarity. (d) Further How required
characterization
of hyper-opalescence
high a concentration in order to suppress
of Cl- would hyper-opalescence
be
Figure 5. The initial rate of super- and hyperopalescence of acto-S-l solutions as a function of the molar ratio of S-l to actin. Various amounts of chymotryptic S-l were added to 2 PM-F-a&in in a solution containing 60 mM-potassium acetate, 20 mMTES, (pH 7.0), 2 mlcr-magnesium acetate, O-2 mm-calcium acetate, 0.1 mM-dithiothreitol, 0.1 mM-NaN,, at 20°C.
completely? As shown in Figure 7, V, of hyperopalescence was sharply reduced by Cl-, while V. of super-opalescence was gradually reduced. About 26 mM of Cl- was sufficient for extinguishing hyperopalescence. Other anions (Br- and NO,) were also tested. These anions inhibited hyper-opalescence with the order of increasing effectiveness of Cl- < Br- < NO;. Why did hyper-opalescence occur around r = l.O? There seem to be two possible interpretations of this observation. One is that actin filaments may acquire an ability to form bundles once they are almost completely decorated with S-l. Another is that free S-l may possess an ability to assemble S-l-decorated actin filaments, for free S-l concentration starts increasing sharply around r = 1.0. is not meant literally. A first Here, “free S-l” interpretation might be that the agent responsible for the actin bundling is S-l that has formed a rigor complex with actin (there cannot be a direct interaction between actin molecules of neighboring filaments). If ADP or .s-ADP inhibit hyperopalescence as well, it may be easy to test which of the interpretations is right. Ranges of the ADP/e-ADP concentrations required for inhibition should differ between the two cases, since the
Acto-S-l Bundling
Figure 6. (a) to (d) Electron micrographs of hyper-opalescence type of acto-S-l 1 PM-F-a&in was incubated at room temperature for 1 h in a solution containing
355
bundles: 1.5 PM-chymotryptic S-l plus 0.1 m-potassium acetate, 20 mM-TES and 0.1 m&x-NaN,, and then mounted both branched filaments point to the
also seem to favor the second interpretation mentioned above. The curve of V,, of hyperopalescence versus the total S-l concentration shifted to the left as ionic strength of the acto-S-l solutions was enhanced, with no significant changes in the plateau value of V, (Fig. 9). As is well known, when ionic strength is enhanced, S-l affinity for actin is reduced. This results in the curve of free S-l concentration versus the total S-l concentration shifting to the left. This fact would be consistent with the shift of V,, if we suppose that free S-l possesses an ability to assemble S-l -decorated actin filaments. In contrast to the effect of TM upon superopalescence, preincubation of F-actin with TM did not significantly affect the V, of hyper-opalescence (Fig. IO). V,, was reduced by 30% by at, most a stoichiometJric amount of TM. This fact suggests also that the molecular process of hyper-opalescence differs from that of super-opalescence.
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1
4-
2-
0 KCI, KBr, KNO, hwl
Figwe 7. Effects of KCl, KBr and KN03 upon the initial rate of hyper-opalescence. F-actin (1 PM) and 2 PMchymotryptic S-l were mxied in a solution containing 20 mM-TES (pH 7.0), 1 mm-magnesium acetate, 0.2 mMcalcium acetate, 0.1 mnn-dithiothreitol, 0.1 mM-NaN,, various
amounts
of one of the neutral salts to be tested
(0, KCI; A, KBr; 0, KNO,), potassium acetate, at 20°C. The sum of potassium acetate concentration and the neutral salts concentration was kept at O-1 M.
Finally, the ability of papain S-l to induce hyperopalescence was examined. Papain S-l also induced hyper-opalescence in essentially the same manner as did chymotryptic S-l (data not shown). 4. Discussion
Tt is known that neutral salts are potent structural destabilizers (at high concentrations) and inhibit the activity of several kinds of enzymes (von Hippel & Wong, 1964; Warren et al,, 1966). The order of increasing effectiveness for monovalent anions is as follows: CH3COO- < Cl- < Br- < NO, < ClO, < I- < SCN-. This order is known as Hofmeister’s series or the lyotropic series. The order of the first four anions coincides with that of their ability to inhibit super- and hyper-opalescence. Stafford (1985) has reported a disruptive effect of Cl- on the myosin tail. Carboxyl ions that exist as organic ions generally comprise 30 to 40% of the intracellular anions. The rest is predominantly composed of phosphate ions. Chloride ion exists only at 1.5 mM in frog sartorius muscle cells. (In spite of these facts, many biochemists have long been using KC1 rather than KCOOCH, to set the ionic strength.) Taking the foregoing facts into consideration, it is natural to assume that Clinhibited an intrinsic ability of S-l to assemble actin filaments, rather than that CH,COO- affects S-l, so that S-l acquires an ability to assemble actin filaments.
ADP, r-ADP
QLLM)
Figure 8. Effect of preincubation of F-actin with ADP or E-ADP on the initial rate of hyper-opalescence and super-opalescence. The data for super-opalescence (0, 0) are the same as those in Fig. 2. (A) Hyper-opalescence rate with ADP; (A) hyper-opalescence rate with E-ADP. Chymotryptic S-l (2 p(M) was added to 1 PM-F-actin preincubated in various amounts of ADP/&-ADP, 0.1 Mpotassium acetate, 20 mM-TES (pH 7*0), 1 mM-magnesium acetate, O-2 mM-calcium acetate, 0.1 mM-dithiothreitol, 0.1 m&i-NaN,, at 20°C. In actin bundles formed in the super-opalescence phase, an S-l molecule seems to cross-link two adjacent actin filaments. Indeed, electron micrographs of the actin bundles show an interfilament distance of 90 A (Ando & Scales, 1985), which is larger than the interfilament distance expected when actin filaments bind directly to each other side by side. When the S-l particle cross-links actin filaments, it must bind tightly to an actin filament at its so-called rigor binding site and must bind weakly to another filament at its secondary binding site. Although ADP binding to acto-S-l weakens the rigor binding, it does not weaken it sufficiently to dissociate S-l from actin. Therefore, the inhibitory effect of ADP on super-opalescence must act by weakening the secondary binding. On the other hand, ADP addition to the super-opalescence type of actin bundles could not disassemble the bundles. There may be two possible interpretations for this. ADP may not be able to bind to S-l that has cross-linked actin filaments. This idea implies that the ADP binding site and the secondary actin binding site may overlap on S-l. Alternatively, there may exist a “locked state” of the bundles, following
an intermediate
bundle
state
that
can
revert to single filaments. The locked state would not revert to the intermediate state even when it attaches to ADP, and ADP-attached single filaments cannot go forward to the intermediate bundle state.
Acto-S-l Bundling
357
12
IO
8 _c1 Ic $
4
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0
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0.04
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Figure 9. Ionic strength effect on the relation of V, versus r. Various amounts of chymotryptic S-l were added to 1 PM-F-a&in in a solution containing various amounts of potassium acetate, 20 mM-TES (pH 7.0), 1 man-magnesium acetate, 0.2 mm-calcium acetate, 0.1 m&r-dithiothreitol, 0.1 mm-NaN,, at 20°C. Potassium acetate wasat (0) 60 mM; (A), 0.1 M; (O), 0.2 M. Although the maximum initial rate of hyperopalescencewas 14 times greater than that of superopalescence, this does not mean that the corresponding rates of the two types of bundling processesdiffer by this much; a unit change in lightscattering intensity accompanied by dimerization of fully S-l-decorated filaments must be much larger than that accompanied by dimerization of partially S- 1-decorated filaments. It should be mentioned here that a few preparations of chymotryptic S-l and papain S-l did not show hyper-opalescence. Even such S-l complexes, however, always showed super-opalescence and normal Ca’+- and EDTA-ATPase activities. This subtle change in S-l quality occurred mainly in the lyophilization process. Aging of S-l solutions also reduced the rate of hyperopalescence. Since further addition of fresh dithiothreitol solution somewhat restored S-l that had not shown normal hyper-opalescence, oxidation of S-l seemsto be responsible for the subtle change in S-l behavior. Seymour & O’Brien (1985) have observed, with an electron microscope, well-ordered acto-S-l bundles formed in a crack in the carbon film. They have mentioned that adjacent filaments in the bundles had antiparallel polarity and spacing of around 150 A. It is not clear whether the bundles they observed are the same as those observed here, because they could not know the solution condi-
Figure 10. Effect of TM on the initial rate of hyperopalescence.Chymotryptic S-l (2 FM) was addedto 1 pmF-actin plus various amounts of TM in a solution containing 0.1 M potassiumacetate, 20 mM-TES (pH 7.0), 1 mM-magnesium acetate, 0.2 m&r-calcium acetate, 0.1 mm-dithiothreitol, 0.1 mM-NaN,, at 20°C. tions (e.g. molar ratio of S-l to actin, ionic condition) in the crack of the carbon film where the acto-S-l bundles were formed. Although we saw a parallel polarity of adjacent filaments in a few specimens of bundles whose filaments branched at an end, it may be too early to generalize this observation to all hyper-opalescence types of bundle. This issue could be resolved in future structural studies on the complex. Hyper-opalescence-type acto-S-l bundles have an interfilament distance of 180 A. This distance is twice that of the super-opalescence-type bundles. Therefore, it is unlikely that an S-l molecule fills this interfilament gap by itself. That is, the S-l molecule is not in contact with both of the neighboring actin filaments. Hyper-opalescence scarcely occurred when r was less than unity. It occurred abruptly when S-l concentration exceeded the value of the actin concentration. Furthermore, the inhibitory effect of ADP on hyper-opalescence appeared in a range where the ADP concentration was much lower than the dissociation constant of ADP for acto-S-l. That is, in spite of the small amount of ADP bound to acto-S-l (at most only 2 to 3 o/o of actinbound S- 1 contains ADP), formation of the hyper-opalescence type of bundles was inhibited by ADP addition. Therefore, ADP acted on S-l that was not involved in rigor complexes with actin (we usually call such S-l “free S-l”), which resulted in inhibition of bundle formation. Therefore, data of the inhibitory effect of ADP suggested that “free S-l ” induced the hyper-opalescence type of acto-S- 1 bundling. This
358
T. Ando
idea can give a clear answer to the question of why hyper-opalescence occurred abruptly when S-l concentration exceeded actin concentration. The “free S-l” concentration is small before the S-l concentration reaches the same value as the actin concentration, but sharply increases after that. This idea is also consistent with the data on ionic strength effects on hyper-opalescence behavior as a function of S-l concentration. “Free S-l” can be obtained to some extent even before S-l concentration reaches the level of the actin concentration; this is achieved by reducing S-l rigor affinity for actin by increasing the ionic strength of the actoS-l solutions. Therefore, in higher ionic strength solutions, hyper-opalescence appeared before the S-l concentration reached the level of the actin concentration. Because these three facts concerning hyper-opalescence are consistent with each other, it is very probable that “free S-l” somehow bundles S- 1-decorated actin filaments. The next question will be how “free S-l ” bundles acto-S-l filaments. It is uncertain at present whether the “free S-l” is incorporated into the final structure of the acto-S-l bundles. It might be possible that the “free S-l” is incorporated into a transient filament-filament complex, from which it is eventually expelled. Whether this is the case or not, it is evident that the “free S-l” interacts with actin-bound S-l. Studies by Morel’s group (Morel & Garrigos, 1982; Bachouchi et al., 1985) have shown that S-l molecules can dimerize through end-to-end contact. According to these authors the dimer is predominant at low ionic strength, but, when the ionic strength is raised using KC1 (approx. 0.1 M), the dimer is disassembled. It would be interesting to see whether such S-l dimers could be retained when KCOOCH, is used instead of KCl, because hyper-opalescence occurred with KCOOCH, but not with KCl. Edited
I thank MS Mei Lee Wong for excellent technical assistance in taking electron micrographs. This work was supported by grants NSF PCM75-22698 and NHBLI- 16683. References Ando, T. (1984). Biochemistry, 23, 375-381. Ando, T. & Asai, H. (1979). J. Mol. Biol. 129, 265-277. Ando, T.&Scales, D. (1985). J. Biol. Chem. 260,2321-2327. Bachouchi, N., Gilik, A., Garrigos, M. & Morel. J. E. (1985). Biochemistry, 24, 6305-6310. Bailey, K. (1948). Biochem. J. 43, 271-279. Ebashi, S., Wakabayashi, T. & Ebashi, F. (1971). J. Biochem. 69, 441-445. Greene, L. E. & Eisenberg, E. (1980). J. Biol. Chem. 255, 5433548. Highsmith, S. (1976). J. BioE. Chem. 251, 6170-6172. Konrad, M. & Goody. R. S. (1982). Eur. J. Biochem. 128, 547-555. Korn. E. D. (1982). Physiol. Rev. 62, 672-737. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265-275. Morel, J. E. & Garrigos, M. (1982). Biochemistry, 21, 2979-2986. Seymour, J. & O’Brien, E. J. (1985). J. Muscle Res. Cell Motil. 6, 725-756. Sheterline, P. (1983). In Mechanism of Cell MotiZity: Molecular Aspects of Contractility, pp. 104-196, Academic Press, New York. Spudich, J. A. & Watt, S. (1971). J. BioZ. Chem. 246, 4866-487 1. Stafford, W. F. (1985). Biochemistry, 24, 3314-3321. Tonomura, Y., Appel, P. & Morales, M. F. (1966). Biochemistry, 5, 515-521. Trinick, J. & Offer, G. (1979). J. Mol. Biol. 133, 549-556. von Hippel, P. H. & Wong, K. Y. (1964). Science, 145, 577-580. Warren, J. C., Stowring, L. & Morales, M. F. (1966). J. Biol. Chem. 241, 309-316. Weeds, A. G. (1982). Nature (London), 296, 811-816. Weeds, A. G. & Taylor, R. S. (1975). Nature (London), 257, 5456. Yanagida, T. (1981). J. Mol. Biol. 146, 539-560.
by H. E. Huxley
Bundling
of Myosin Subfragment-l-decorated Actin Filaments Toshio Andot Cardiovascular Research Institute University of California San Francisco, CA 94143, IJ.S.A.
(Received 29 July 1986, and in revised form
2 January
1987)
We have reported previously that rabbit skeletal myosin subfragment-l (S-l) assembles actin filaments into bundles. The rate of this reaction can be estimated roughly from the initial rate (V,) of the accompanying turbidity increase (“super-opalescence”) of the acto-S-l solution. V, is a function of the molar ratio (r) of S-l to actin, with a peak at r = l/S to l/7 and minimum around r = 1. In the present paper we report a different type of opalescence (we call it “hyperopalescence”) of a&o-S-l solutions, which also resulted from bundle formation. Adjacent filaments in the bundles had a distance of approximately 180 8. Hyper-opalescence occurred at r x 1 when KCOOCH, was used instead of KCl. By comparing the effects of ADP, .+ADP, tropomyosin or ionic strength upon the super- and hyper-opalescence, we concluded that the two types of S-l-induced actin bundling had different molecular mechanisms. The hyper-opalescence type of bundling seemedto be induced by S-l, which was not complexed with actin in the manner of conventional rigor binding. The presence of the regulatory light chain did not affect hyper-opalescence (or super-opalescence), since there were no significant differences between papain S-l and chymotryptic S-l with respect to these phenomena.
is about 90 A. Initially, they observed that, when an appropriate amount of S-l was added to F-actin Actin is ubiquitous in eukaryotic cells. In nonsolutions, an instant enhancement of lightmuscle cells, actin exists in a variety of forms (i.e. scattering (due to S-l binding to F-actin) was monomer, filament, bundle, and mesh-work strucfollowed by a gradual increase of the scattering ture) and can change its form when required. with time. This gradual increase was called “superAssembly of actin filaments into higher-order opalescence”. The initial rate of super-opalescence structures is assisted by a variety of actin-binding was not proportional to the amount of S-l added, proteins such as fascin, fimbrin, villin, filamin, and but had a peak at an S-l to actin molar ratio (r) of a protein identified in lung macrophages (for l/S to l/7. The rate was nearly zero at r values reviews, see Weeds, 1982; Korn, 1982; Sheterline, above 0.5. In the course of the experiments, it’ was 1983). Heavy meromyosin prepared from skeletal noticed that the rate of super-opalescence was muscle myosin cross-links actin filaments in vitro by enhanced drastically, simply by replacing KC1 with binding each S-l portion to a different actin KCOOCH3. Not only was the magnitude changed, filament to form a raft-like structure (Trinick & but so also was the behavior of the rate as a Offer, 1979). It has been found by Ando & Scales function of r. Once r approached 1, the light-scattering (1985) that even the single-headed subfragment of intensity of the acto-S-l solutions increased rabbit skeletal myosin, S-1$, can assemble actin abruptly. Since this behavior is so peculiar, we can filaments into bundles whose interfilament distance expect that some unknown interactions between the proteins must be involved. In order to investigate t Present. address: Department of Physics, Faculty of such interactions, we have characterized acto-S-l Science, Kanazawa University, 1-l Marunouchi, solutions that contain acetate as a predominant Kanazawa, Ishikawa 920 Japan. anion, by studying the effects of nucleotides, $ Abbreviations used: myosin S-l, subfragment- 1; tropomyosin (TM), and ionic strength on the lightTM. tropomyosin; TES, N-tris[hydroxymethyl]methylscattering behavior, and by observing acto-S-l with 2-aminoethane sulfonic acid; E-ADP, 1 ,N6an electron microscope. ethenoadenosine 5’-diphosphate. 351 1. Introduction
OO22-2836/87/100351-08
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Press
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(London)
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352
T. Ando according to the methods of Bailey (1948) and Ebashi et al. (1971), respectively. The molar concentrations of papain S-l, chymotryptic S-l and F-actin were determined by their ultraviolet absorption in 0.6 M-NaCl using E&& = 8.3, Ei& = 7.5 and Ei& = 65, respectively. and respective molecular weights of 1.33 x 105, 1.15 x lo5 and 4.2 x 104. Corrections for turbidity were made as described (Ando, 1984). The molar concentrations of TM and troponin were determined by the method of Lowry et al. (1951), taking values of 7.0 x lo4 and 7.5 x IO4 for their respective molecular weights. (b) Light-scattering
20
40
60
I 80
I
KCI, KEr. KNO, hd
Figure 1. Effects of KCl, KBr and KNO, upon the initial rate (V,) of super-opalescence. F-actin (3 PM) and chymotryptic S-l (0.45pM) were mixed in a solution containing 20 mM-TES (pH 7.0), 1 ITIM-UU3gneSiUm acetate, 0.2 m&r-calcium acetate, 0.1 mxl-dithiothreitol, 0.1 mM-KaKs and various amounts of one of the neutral salts to be tested (0, KCl; A, KBr; 0, KNO,) and potassium acetate at 20°C. The sum of potassium acetate concentration and the neutral salts concentration was kept at 0.1 M.
As described by Ando & Scales (1985), the lightscattering intensity of acto-S-l solutions was measured at 400 nm using a Hitachi Perkin-Elmer MPF4 fluorometer. Since the units of light-scattering intensity are arbitrary, the intensity was normalized by assigning a value of 10 to the intensity of 5 p&r-F-actin in a solution containing 0.1 M-KCl, 20 miw-TES (pH 7.0), 2 mM0.2 miw-Ca(CGGCH,),, 0.2 mm-dithioMg(COOCH,),, threitol and 0.1 mM-NaN, at 15°C. A small volume (5 to 160 ~1) of S-l solution was added to 2 ml of F-actin solution in a thermostatted fluorescence cuvette. The solution was mixed by sucking and blowing several times with a l-ml Pipetman automatic pipet, and the timecourse of changes in light-scattering intensity was immediately recorded on time-scanning chart paper. The initial rate (V,) of super-opalescence or hyper-opalescence was read from the chart paper. (c) Electron microscopy
2. Materials and Methods (a) Protein preparations Myosin was prepared from rabbit skeletal back and leg muscles (Tonomura et al., 1966) and stored in 0.3 M-KCl, 50% (v/v) glycerol at -20°C. Papain S-l was obtained by digesting myosin with insoluble papain in a solution containing 0.1 M-ammonium acetate (pH 7.2), 2 mMMgCl,, and 0.2 miw-dithiothreitol, and purified by the use of DEAE-Sepharose CL-6B. The purified S-l was concentrated using Sephadex G-25 powder. Chymotryptic S-l was obtained by the method of Weeds & Taylor (1975). S-l(A1) and Sl(A2) were separated using preparative DEAE-cellulose high-pressure liquid chromatography (h.p.1.c.). The eluted S-l was concentrated by 60% saturation of ammonium sulfate. After removal of the ammonium sulfate, the concentrated S-l was lyophilized in 0.15 M-SUCrOSe, 50 tIIMn-a~IUOniUIn acetate and 05 mw-dithiothreitol at pH 7.0. The lyophilized S-l was stored at -20°C. Just before use, the lyophilized S-l was dissolved with a small volume of the desired buffer solution, and clarified by centrifuging for 90 min at 47,000 revs/min using a 50 rotor. The h.p.1.c. and lyophilization steps were omitted in some cases. However, this omission did not significantly affect the results given below. Acetone powder of rabbit skeletal muscle was prepared as described (Ando & Asai, 1979). Actin was first purified by the method of Spudich & Watt (1971), and further purified by Sephacryl S-300 gel chromatography. A high concentration (15 mg/ml) of F-actin was stored in 0.1 mmATP, 20 mM-KCOOCH,, 20 miw-TES (pH 7.0). 2 mM-Mg(COOCH,),, 0.2 mMCa(COOCH,),, 0.2 m&r-dithiothreitol, 0.1 mmNaru’, at 6°C. Just before use, the stored F-actin was diluted to approx. 20 pM with an ATP-free buffer solution. TM and troponin were prepared from rabbit skeletal muscle
Droplets of solutions of 1 mw-F-actin plus 1.5 PM-S-~ were applied to freshly carbon-coated Formvar grids. After 30 s, a few drops of 1 ye (w/v) uranyl acetate were applied to the grid for another 30 s. The negative stain was then removed with torn filter paper. The grids were allowed to dry for several minutes before they were observed at 80 kV in a Philips EM 200 electron microscope.
3. Results (a) Further
characterization
of super-opalescence
When super-opalescence of acto-S-l solutions was first studied (Ando & Scales, 1985), KC1 was used for setting an appropriate ionic strength. Here, however, Cl- was found to decelerate super-opalescence (see Fig. 1). F-actin (3 ,UM) and chymotryptic S-l (0.45 PM) (i.e. r = 0.15) were used in this experiment. The sum of KCi and KCOOCH, concentrations was kept at 0.1 M. V, with 0.1 M-KCOOCH, was about 15-fold higher than that with 0.1 M-KCl. Therefore, unless otherwise mentioned, KCOOCH3 was used instead of KCl. The inhibitory effect of and NO;) was also tested. As other anions (Brshown in Figure 1, these anions inhibited superopalescence with the order of increasing effectiveness being Cl- < Br- < NO;. V, decreased when ADP had been preincubated in F-a&in solutions before adding chymotryptic S-l. V, decreased to zero with increasing ADP concentration (Fig. 2). V,, decreased by half at approximately 20 PM-ADP. This value is not very
Acto-S-l
353
Bundling
r0
F14
\ 0 0
Ej-
\
0
T .E .E 2
4
) i
\ ‘\,
\0 ‘0
\@, \ ,O\” C
I
I
0.2
I
I
0.4 ADP, E -ADP
0.6
I
C
(mtd
Figure 2. Effect of preincubation of F-actin with ADP or E-ADP on the initial rate of super-opalescence. Chymotryptic S-l (0.45 PM) was added to 3 pni-F-actin amounts of ADP/e-ADP, preincubated in various 60 mM-potassium acetate, 20 mM-TES (PH 74L acetate, 0.2 mM-calcium acetate, 2 mM-magnesium 0.1 mw-dithiothreitol, and 0.1 mM-NaNu’,, at 20°C. 0. ADP effect; 0, E-ADP effect.
different from the reported values of the dissociation constant of Mg 2+ -ADP for rabbit skeletal actoS-l: 37 PM (Highsmith, 1976), 143~~ (Greene & Eisenberg, 1980). When E-ADP was used instead of ADP,
V, decreased
by
half
at
approximately
270 PM-E-ADP. This value is also not far from the dissociation constant of Mg’+-&-ADP (350 /AM) for rabbit skeletal acto-myosin in fibers reported by Yanagida (1981). Therefore, the acto-S-l-bound nucleotides seem to be responsible for the reduction of V,. This conclusion is plausible, considering that V, is not, a linear function of the amount of nucleotide-free acto-S-l complex. On the other hand, when ADP was added to a&o-S-l solutions that had been preincubated and were already showing super-opalescence, the lightscattering intensity stayed at a level slightly lower than just before ADP was added (Fig. 3). Since mechanical perturbation alone of acto-S-l solutions by mixing with a Pipetman reduced the superopalescencelevel, the slight reduction observed here was not due to ADP addition. Therefore, we can conclude that ADP addition to actin bundles cannot disassemble the bundles, although ADP inhibits bundle formation. TM had similar effects on the super-opalescence of acto-S-l solutions. TM addition to actin bundles cannot disassemble the bundles whereas TM inhibits bundle formation. A molar ratio of TM/a&in of as little as approximately 0.018 was sufficient to reduce V, by half (Fig. 4). Troponin had little influence on the TM effects on super-
0
I 2
I 4
I 6
I 8
I IO
I I2
I 14
Time (mini
Figure 3. Effect on the super-opalescence of ADP addition to the acto-S-l solution that has shown superopalescence. At arrow 1, 0.45 PM-chymotryptir S-l was added to 3 PM-F-actin in a solution containing 0.1 M-potassium acetate, 20 mM-TES (pH 7.0), 1 mMmagnesium acetate, 0.2 mM-calcium acetate, 0.1 mMdithiothreitol, 0.1 mM-NaN,, at 20°C. At arrow 2. 0.2 mM-ADP was added.
opalescence, irrespective of the presence of Ca’+. To study the effect of the regulatory light chain on super-opalescence, the previous experiments were repeated using papain-S-1 instead of chymotryptic S-l. It gave essentially the same results as those obtained with chymotryptic S-I. (b) Hyper-opalescence We have reported (Ando & Scales, 1985) that V, reaches a peak at r = l/S to l/7, and then steadily decreasesto nearly zero when r is further increased (see Figs 2 and 3 of Ando & Scales, 1985). Though V, data at r above 0.7 were not shown in that paper, V, was zero at this range of r values (Fig. 3 of Ando & Scales (1985) implies this fact). Now, V, versus r was re-studied in solutions that did not contain Cl-, but contained CH,COO- instead. As shown in Figure 5 (chymotryptic S-l was used), V, reached a peak at r = l/6 to l/7 and then decreased to zero when r was increased (the first phase). However, as r was further increased, V, st#arted increasing very rapidly just’ before r reached the value 1.0, and reached a plateau at r z 2.5 (the second phase). The plateau value of V,, at r z 2.5 was about 14 times larger than the peak value of the first phase. The contrast between the present and previous observations of V, behavior around r = 1.0 resulted only from the difference of anion (i.e. Cl- or CH,COO-). Henceforth, in order to
354
T. Ando
I
I
I
I
I
I
0.08
0.04
0
I
0.12
0.16
TM Q.LM)
Figure 4. Effect of preincubation of F-actin with TM on the initial rate of super-opalescence. Chymotryptic S-l (0.45 PM) was added to 3 PM-F-a&in plus various amounts of TM in a solution containing 60 mm-potassium acetate, 20 mM-TES (pH 7.0), 2 m&r-magnesium acetate, 0.2 mm-calcium acetate, 0.1 m&r-dithiothreitol, O-1 mMNaN,, at 20°C. distinguish the second phase from the first phase, we call the second one “hyper-opalescence” and we use “super-opalescence” for the first one. (c) Bundle
formation
Does hyper-opalescence also result from formation of bundles by S-l-decorated F-actin? In order to study this question acto-S-l was observed with an electron microscope. An acto-S-l sample (1.5 PMchymotryptic S-l plus 1 pi%-F-actin) was incubated at room temperature for one hour in a solution containing 0.1 M-KCOOCH,, 20 mM-TES (pH 7.0), 1 miw-Mg(COOCH,),, 0.2 miw-Ca(COOCH,),, 0.2 m&r-dithiothreitol, O-1 mM-NaN,, and then mounted on grids. As shown in Figure 6(a), wellordered bundles containing a few S-l-decorated actin filaments were observed. The bundles possessed distinct transverse stripes with a periodicity of about 350 A and an interfilament distance of about 180 A. It was difficult to see any arrowhead polarity of an S-l-decorated filament within the bundles. Fortunately, we had a few specimens of bundles in which two adjacent filaments branched at an end, so that we could clearly see the polarity (Fig. 6(b), (c), (d)). The arrowheads of both branched filaments pointed in the same direction (i.e. to the junction ((b), (c)), or to the splay direction (d)). That is, actin filaments within these particular specimens had parallel polarity. (d) Further How required
characterization
of hyper-opalescence
high a concentration in order to suppress
of Cl- would hyper-opalescence
be
Figure 5. The initial rate of super- and hyperopalescence of acto-S-l solutions as a function of the molar ratio of S-l to actin. Various amounts of chymotryptic S-l were added to 2 PM-F-a&in in a solution containing 60 mM-potassium acetate, 20 mMTES, (pH 7.0), 2 mlcr-magnesium acetate, O-2 mm-calcium acetate, 0.1 mM-dithiothreitol, 0.1 mM-NaN,, at 20°C.
completely? As shown in Figure 7, V, of hyperopalescence was sharply reduced by Cl-, while V. of super-opalescence was gradually reduced. About 26 mM of Cl- was sufficient for extinguishing hyperopalescence. Other anions (Br- and NO,) were also tested. These anions inhibited hyper-opalescence with the order of increasing effectiveness of Cl- < Br- < NO;. Why did hyper-opalescence occur around r = l.O? There seem to be two possible interpretations of this observation. One is that actin filaments may acquire an ability to form bundles once they are almost completely decorated with S-l. Another is that free S-l may possess an ability to assemble S-l-decorated actin filaments, for free S-l concentration starts increasing sharply around r = 1.0. is not meant literally. A first Here, “free S-l” interpretation might be that the agent responsible for the actin bundling is S-l that has formed a rigor complex with actin (there cannot be a direct interaction between actin molecules of neighboring filaments). If ADP or .s-ADP inhibit hyperopalescence as well, it may be easy to test which of the interpretations is right. Ranges of the ADP/e-ADP concentrations required for inhibition should differ between the two cases, since the
Acto-S-l Bundling
Figure 6. (a) to (d) Electron micrographs of hyper-opalescence type of acto-S-l 1 PM-F-a&in was incubated at room temperature for 1 h in a solution containing
355
bundles: 1.5 PM-chymotryptic S-l plus 0.1 m-potassium acetate, 20 mM-TES and 0.1 m&x-NaN,, and then mounted both branched filaments point to the
also seem to favor the second interpretation mentioned above. The curve of V,, of hyperopalescence versus the total S-l concentration shifted to the left as ionic strength of the acto-S-l solutions was enhanced, with no significant changes in the plateau value of V, (Fig. 9). As is well known, when ionic strength is enhanced, S-l affinity for actin is reduced. This results in the curve of free S-l concentration versus the total S-l concentration shifting to the left. This fact would be consistent with the shift of V,, if we suppose that free S-l possesses an ability to assemble S-l -decorated actin filaments. In contrast to the effect of TM upon superopalescence, preincubation of F-actin with TM did not significantly affect the V, of hyper-opalescence (Fig. IO). V,, was reduced by 30% by at, most a stoichiometJric amount of TM. This fact suggests also that the molecular process of hyper-opalescence differs from that of super-opalescence.
356
T. Ando
1
4-
2-
0 KCI, KBr, KNO, hwl
Figwe 7. Effects of KCl, KBr and KN03 upon the initial rate of hyper-opalescence. F-actin (1 PM) and 2 PMchymotryptic S-l were mxied in a solution containing 20 mM-TES (pH 7.0), 1 mm-magnesium acetate, 0.2 mMcalcium acetate, 0.1 mnn-dithiothreitol, 0.1 mM-NaN,, various
amounts
of one of the neutral salts to be tested
(0, KCI; A, KBr; 0, KNO,), potassium acetate, at 20°C. The sum of potassium acetate concentration and the neutral salts concentration was kept at O-1 M.
Finally, the ability of papain S-l to induce hyperopalescence was examined. Papain S-l also induced hyper-opalescence in essentially the same manner as did chymotryptic S-l (data not shown). 4. Discussion
Tt is known that neutral salts are potent structural destabilizers (at high concentrations) and inhibit the activity of several kinds of enzymes (von Hippel & Wong, 1964; Warren et al,, 1966). The order of increasing effectiveness for monovalent anions is as follows: CH3COO- < Cl- < Br- < NO, < ClO, < I- < SCN-. This order is known as Hofmeister’s series or the lyotropic series. The order of the first four anions coincides with that of their ability to inhibit super- and hyper-opalescence. Stafford (1985) has reported a disruptive effect of Cl- on the myosin tail. Carboxyl ions that exist as organic ions generally comprise 30 to 40% of the intracellular anions. The rest is predominantly composed of phosphate ions. Chloride ion exists only at 1.5 mM in frog sartorius muscle cells. (In spite of these facts, many biochemists have long been using KC1 rather than KCOOCH, to set the ionic strength.) Taking the foregoing facts into consideration, it is natural to assume that Clinhibited an intrinsic ability of S-l to assemble actin filaments, rather than that CH,COO- affects S-l, so that S-l acquires an ability to assemble actin filaments.
ADP, r-ADP
QLLM)
Figure 8. Effect of preincubation of F-actin with ADP or E-ADP on the initial rate of hyper-opalescence and super-opalescence. The data for super-opalescence (0, 0) are the same as those in Fig. 2. (A) Hyper-opalescence rate with ADP; (A) hyper-opalescence rate with E-ADP. Chymotryptic S-l (2 p(M) was added to 1 PM-F-actin preincubated in various amounts of ADP/&-ADP, 0.1 Mpotassium acetate, 20 mM-TES (pH 7*0), 1 mM-magnesium acetate, O-2 mM-calcium acetate, 0.1 mM-dithiothreitol, 0.1 m&i-NaN,, at 20°C. In actin bundles formed in the super-opalescence phase, an S-l molecule seems to cross-link two adjacent actin filaments. Indeed, electron micrographs of the actin bundles show an interfilament distance of 90 A (Ando & Scales, 1985), which is larger than the interfilament distance expected when actin filaments bind directly to each other side by side. When the S-l particle cross-links actin filaments, it must bind tightly to an actin filament at its so-called rigor binding site and must bind weakly to another filament at its secondary binding site. Although ADP binding to acto-S-l weakens the rigor binding, it does not weaken it sufficiently to dissociate S-l from actin. Therefore, the inhibitory effect of ADP on super-opalescence must act by weakening the secondary binding. On the other hand, ADP addition to the super-opalescence type of actin bundles could not disassemble the bundles. There may be two possible interpretations for this. ADP may not be able to bind to S-l that has cross-linked actin filaments. This idea implies that the ADP binding site and the secondary actin binding site may overlap on S-l. Alternatively, there may exist a “locked state” of the bundles, following
an intermediate
bundle
state
that
can
revert to single filaments. The locked state would not revert to the intermediate state even when it attaches to ADP, and ADP-attached single filaments cannot go forward to the intermediate bundle state.
Acto-S-l Bundling
357
12
IO
8 _c1 Ic $
4
2
0
I
I
I
0.04
0.08
0.14
TM (prd
r(S-i/actin)
Figure 9. Ionic strength effect on the relation of V, versus r. Various amounts of chymotryptic S-l were added to 1 PM-F-a&in in a solution containing various amounts of potassium acetate, 20 mM-TES (pH 7.0), 1 man-magnesium acetate, 0.2 mm-calcium acetate, 0.1 m&r-dithiothreitol, 0.1 mm-NaN,, at 20°C. Potassium acetate wasat (0) 60 mM; (A), 0.1 M; (O), 0.2 M. Although the maximum initial rate of hyperopalescencewas 14 times greater than that of superopalescence, this does not mean that the corresponding rates of the two types of bundling processesdiffer by this much; a unit change in lightscattering intensity accompanied by dimerization of fully S-l-decorated filaments must be much larger than that accompanied by dimerization of partially S- 1-decorated filaments. It should be mentioned here that a few preparations of chymotryptic S-l and papain S-l did not show hyper-opalescence. Even such S-l complexes, however, always showed super-opalescence and normal Ca’+- and EDTA-ATPase activities. This subtle change in S-l quality occurred mainly in the lyophilization process. Aging of S-l solutions also reduced the rate of hyperopalescence. Since further addition of fresh dithiothreitol solution somewhat restored S-l that had not shown normal hyper-opalescence, oxidation of S-l seemsto be responsible for the subtle change in S-l behavior. Seymour & O’Brien (1985) have observed, with an electron microscope, well-ordered acto-S-l bundles formed in a crack in the carbon film. They have mentioned that adjacent filaments in the bundles had antiparallel polarity and spacing of around 150 A. It is not clear whether the bundles they observed are the same as those observed here, because they could not know the solution condi-
Figure 10. Effect of TM on the initial rate of hyperopalescence.Chymotryptic S-l (2 FM) was addedto 1 pmF-actin plus various amounts of TM in a solution containing 0.1 M potassiumacetate, 20 mM-TES (pH 7.0), 1 mM-magnesium acetate, 0.2 m&r-calcium acetate, 0.1 mm-dithiothreitol, 0.1 mM-NaN,, at 20°C. tions (e.g. molar ratio of S-l to actin, ionic condition) in the crack of the carbon film where the acto-S-l bundles were formed. Although we saw a parallel polarity of adjacent filaments in a few specimens of bundles whose filaments branched at an end, it may be too early to generalize this observation to all hyper-opalescence types of bundle. This issue could be resolved in future structural studies on the complex. Hyper-opalescence-type acto-S-l bundles have an interfilament distance of 180 A. This distance is twice that of the super-opalescence-type bundles. Therefore, it is unlikely that an S-l molecule fills this interfilament gap by itself. That is, the S-l molecule is not in contact with both of the neighboring actin filaments. Hyper-opalescence scarcely occurred when r was less than unity. It occurred abruptly when S-l concentration exceeded the value of the actin concentration. Furthermore, the inhibitory effect of ADP on hyper-opalescence appeared in a range where the ADP concentration was much lower than the dissociation constant of ADP for acto-S-l. That is, in spite of the small amount of ADP bound to acto-S-l (at most only 2 to 3 o/o of actinbound S- 1 contains ADP), formation of the hyper-opalescence type of bundles was inhibited by ADP addition. Therefore, ADP acted on S-l that was not involved in rigor complexes with actin (we usually call such S-l “free S-l”), which resulted in inhibition of bundle formation. Therefore, data of the inhibitory effect of ADP suggested that “free S-l ” induced the hyper-opalescence type of acto-S- 1 bundling. This
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idea can give a clear answer to the question of why hyper-opalescence occurred abruptly when S-l concentration exceeded actin concentration. The “free S-l” concentration is small before the S-l concentration reaches the same value as the actin concentration, but sharply increases after that. This idea is also consistent with the data on ionic strength effects on hyper-opalescence behavior as a function of S-l concentration. “Free S-l” can be obtained to some extent even before S-l concentration reaches the level of the actin concentration; this is achieved by reducing S-l rigor affinity for actin by increasing the ionic strength of the actoS-l solutions. Therefore, in higher ionic strength solutions, hyper-opalescence appeared before the S-l concentration reached the level of the actin concentration. Because these three facts concerning hyper-opalescence are consistent with each other, it is very probable that “free S-l” somehow bundles S- 1-decorated actin filaments. The next question will be how “free S-l ” bundles acto-S-l filaments. It is uncertain at present whether the “free S-l” is incorporated into the final structure of the acto-S-l bundles. It might be possible that the “free S-l” is incorporated into a transient filament-filament complex, from which it is eventually expelled. Whether this is the case or not, it is evident that the “free S-l” interacts with actin-bound S-l. Studies by Morel’s group (Morel & Garrigos, 1982; Bachouchi et al., 1985) have shown that S-l molecules can dimerize through end-to-end contact. According to these authors the dimer is predominant at low ionic strength, but, when the ionic strength is raised using KC1 (approx. 0.1 M), the dimer is disassembled. It would be interesting to see whether such S-l dimers could be retained when KCOOCH, is used instead of KCl, because hyper-opalescence occurred with KCOOCH, but not with KCl. Edited
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by H. E. Huxley