18.6: Actin-Myosin Interactions In Vitro - Dissections and Reconstitutions

Several experiments hinted at the interaction of actin and myosin in contraction. For example, actomyosin was first observed as the main component of viscous skeletal muscle homogenates. Under appropriate conditions, adding ATP to such actomyosin preparations caused a decrease in viscosity. However, after the added ATP was hydrolyzed, the mixture became viscous again. Extraction of the nonviscous preparation (before it recongealed and the ATP was consumed) led to the biochemical separation of the two main substances we now recognize as the actin and myosin (thin and thick) filaments of contraction. What’s more, adding these components back together reconstituted the viscous actomyosin (now renamed actinomyosin). Adding ATP once again to the reconstituted solution eliminated its viscosity. The ATP-dependent viscosity changes of actinomyosin solutions were consistent with an ATP-dependent separation of thick and thin filaments. Do actin and myosin also separate in glycerinated muscles exposed to ATP, allowing them to stretch and relax? This question was answered with the advent of electron microscopy. The purification of skeletal muscle myosin from actin (still attached to Z-lines) is cartooned in Figure 18.17, showing what the separated components looked like in the electron microscope.

Figure 18.17: Overview of an isolation of actin (thin) filaments (still on Z-lines) from myosin (thick) filaments.

Next, after mixing actin Z-line and myosin fractions, electron microscopy of the resulting viscous material revealed thin filaments interdigitating with thick filaments (Figure 18.18).

Figure 18.18: Reconstitution of actin filaments (on Z-lines) with myosin filaments.

As expected, when ATP was added to these extracts, the solution viscosity dropped, and electron microscopy revealed that the myosin and actin filaments had again separated. The two components could again be isolated and separated by centrifugation.

In yet further experiments, actinomyosin preparations could be spread on over an aqueous surface, producing a film on the surface of the water. When ATP was added to the water, the film visibly “contracted,” pulling away from the edges of the vessel, reducing its surface area! Electron microscopy of the film revealed shortened, sarcomere-like structures, with closely spaced Z-lines and short I-bands—further confirming the sliding-filament model of muscle contraction.

332 In Vitro & Electron Microscope Evidence for a Sliding-Filament Model

When actin and myosin were further purified from isolated actinomyosin, the thick myosin rods could be dissociated into large myosin monomers. In fact, at ~599 Kd, myosin monomers are among the largest known proteins. Thus, thick filaments are massive polymers of huge myosin monomers! The molecular structure of myosin (thick) filaments is shown in Figure 18.19.

Figure 18.19: Structure of a skeletal-muscle myosin filament and the myosin monomer. Shown is myosin II, the thick filament that spans both sides of the H zone in a sarcomere (upper). The head-and-tail structure of a myosin monomer is shown in the high-magnification electron micrograph and is illustrated in the cartoon (lower). The myosin monomer is itself a polymer of four polypeptides.

An early observation of isolated mammalian actin filaments was that they had no ATPase activity. We’ve seen isolated myosin preparations do have ATPase activity, but they would catalyze ATP hydrolysis only very slowly compared to intact muscle fibers. Faster ATP hydrolysis occurred only if myosin filaments were mixed with microfilaments (either on or detached from Z-lines). In the electron microscope, isolated myosin protein monomers each appeared to have a double-head and a single-tail region. Biochemical analysis showed that the monomers themselves were composed of the two heavy-chain polypeptides and two pairs of light-chain polypeptides, as shown in the Figure 18.19 illustration. In Figure 18.20, a high magnification, high-resolution electron-micrograph simulation and corresponding drawings illustrate a myosin monomer and its component structures.

Figure 18.20: Purified myosin monomers are digested with enzymes that hydrolyze peptide bonds between specific amino acids. This produces an S1 (head) fragment and a tail fragment with different properties.

Proteolytic enzymes that only hydrolyze peptide linkages—and only between specific amino acids—“cut” the myosin monomers into S1 (head) and tail fragments. Shown in Figure 18.20 are electron micrographs of enzymatic digest fractions separated by ultracentrifugation. The tail fragments are parts of the two-myosin heavy-chain polypeptides. The S1 fragments consist of a pair of light chains and the rest of the heavy chains. On further analysis, the S1 myosin head fraction had a slow ATPase activity, while the tails had none. The slow activity was not an artifact of isolation; mixing the S1 fraction with isolated actin filaments resulted in a higher rate of ATP hydrolysis. Thus, the myosin heads must be ATPases that bind and interact with actin microfilaments.

333-2 Thick Filaments & Myosin Monomer Structure

CHALLENGE

What do you think is going on here? Why the faster catalytic rate when actin and myosin (or their parts) are mixed?

In fact, S1 myosin heads bind directly to actin, decorating the actin with “arrowheads” visible in the electron microscope (see S1 Arrowheads On Muscle Actin). Even intact myosin monomers could decorate muscle actin. These results are consistent with the requirement that myosin must bind to actin to achieve a maximum rate of ATPase activity during contraction. The arrowheads on decorated actin still attached to Z-lines are illustrated in Figure 18.21.

Figure 18.21: Actin decoration by myosin-monomer S1 fragments in a kind of reconstitution experiment shows an opposing polarity of actin filaments on opposite sides of the Z-line.

Note that the “arrowheads” always face in opposite directions on either side of the Z-line. These opposing arrowheads suggest that the actin filaments attached to the two Z-lines of a sarcomere are drawn toward each other along the opposite sides of bipolar myosin rods. This is consistent with sliding filaments that draw Z-lines closer together during skeletal muscle contraction, shortening the sarcomeres. For another look at “arrowheads” and other aspects of muscle structure, check out the slide show at Muscle Structure & Physiology-J. Rosenbluth.

334-2 Myosin Monomers & S1 Heads Decorate Actin

CHALLENGE

Explain how the phenomenon of “arrowhead decoration” of sarcomeres can be interpreted to indicate actin “polarity.”