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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 18.3Myosin: The Actin Motor Protein

Although cells can harness polymerization of actin to generate some forms of movement, many cellular movements depend on interactions between actin filaments and myosin, an ATPase that moves along actin filaments by coupling the hydrolysis of ATP to conformational changes. This type of enzyme, which converts chemical energy into mechanical energy, is called a mechanochemical enzyme or, colloquially, a motor protein. Myosin is the motor, actin filaments are the tracks along which myosin moves, and ATP is the fuel that powers movement.

In this section we describe the structure and functions of the three major myosin classes and examine the mechanism of myosin-dependent movement. Contraction, a special form of movement resulting from actin and myosin interactions, is highly evolved in muscle cells, which we consider in the next section. Muscle contraction provides a basis for our subsequent discussion of contractile events involving less organized systems in nonmuscle cells. In the next chapter, we will discuss the microtubule motors kinesin and dynein, which exhibit many of the same properties as myosin.

All Myosins Have Head, Neck, and Tail Domains with Distinct Functions

Thirteen members of the myosin gene family have been identified by genomic analysis (Chapter 7). Myosin I and myosin II, the most abundant and thoroughly studied of the myosin proteins, are present in nearly all eukaryotic cells. A less-common isoform, myosin V, also has been isolated and characterized. Although the specific activities of these myosins differ, they all function as motor proteins. Myosin II powers muscle contraction and cytokinesis, whereas myosins I and V are involved in cytoskeleton-membrane interactions such as the transport of membrane vesicles. The activities of the remaining proteins encoded by the myosin gene family are now being discovered.

All myosins are composed of one or two heavy chains and several light chains. The heavy chains are organized into three structurally and functionally different domains (Figure 18-20a). The globular head domain contains actin- and ATP-binding sites and is responsible for generating force; this is the most conserved region among the various myosins. Adjacent to the head domain lies the α-helical neck region, which is associated with the light chains. The latter regulate the activity of the head domain. The tail domain contains the binding sites that determine the specific activities of a particular myosin, as discussed below.

Figure 18-20. Structure of various myosin molecules.

Figure 18-20

Structure of various myosin molecules. (a) The three major myosin proteins are organized into head, neck, and tail domains, which carry out different functions. The head domain binds actin and has ATPase activity. The light chains, bound to the neck domain, (more...)

Myosin II and myosin V are dimers in which α-helical sequences in the tail of each heavy chain associate to form a rodlike coiled-coil structure. Because the myosin I heavy chain lacks this α-helical sequence, the molecule is a monomer. The three myosins differ in the number and type of light chains bound in the neck region (see Figure 18-20a). The light chains of myosin I and myosin V are calmodulin, a Ca2+-binding regulatory subunit in many intracellular enzymes. Myosin II contains two different light chains (called essential and regulatory light chains); both are Ca2+-binding proteins but differ from calmodulin in their Ca2+-binding properties. All myosins are regulated in some way by Ca2+; however, because of the differences in their light chains, the different myosins exhibit different responses to Ca2+ signals in the cell.

The head, neck, and tail domains of the myosins are clearly visible in electron micrographs. This domain organization has been confirmed by proteolytic studies of myosin II, outlined in Figure 18-20b. The head domain corresponds to the N-terminal half of the heavy chain, and the tail domain to the C-terminal half.

Studies of the myosin fragments produced by proteolysis revealed the biochemical properties of the three domains. In all myosins, the head domain is a specialized ATPase that is able to couple the hydrolysis of ATP with motion. A critical feature of the myosin ATPase activity is that it is actin-activated. In the absence of actin, solutions of myosin slowly convert ATP into ADP and phosphate. However, when myosin is complexed with actin, the rate of myosin ATPase activity is four to five times faster than in the absence of actin. The actin-activation step ensures that the myosin ATPase operates at its maximal rate only when the myosin head domain is bound to actin.

Although the head domain of all myosins exerts force on actin, the role of a particular myosin in cells is related to its tail domain. Because the tail domains of myosin I and myosin V bind the plasma membrane or the membranes of intracellular organelles, these molecules have membranerelated activities (Figure 18-21a). For example, myosin I serves as a linkage between the plasma membrane and the microfilament bundles in brush-border microvilli and in filopodia. In contrast, the rodlike tail domains of multiple myosin II dimers associate to form thick filaments, which compose part of the contractile apparatus in muscle. A thick filament from skeletal muscle has a bipolar organization — the heads are located at both ends of the filament and are separated by a central bare zone devoid of heads (Figure 18-21b). When packed tightly together in a thick filament, many myosin head domains can interact simultaneously with actin filaments.

Figure 18-21. Functions of the myosin tail domain.

Figure 18-21

Functions of the myosin tail domain. (a) Myosin I and myosin V are localized to cellular membranes by undetermined sites in their tail domains. As a result, these myosins are associated with intracellular membrane vesicles or the cytoplasmic face of the (more...)

Myosin Heads Walk along Actin Filaments

Studies of muscle contraction provided the first evidence that myosin heads slide or walk along actin filaments. Unraveling the mechanism of muscle contraction was greatly aided by development of in vitro motility assays that permit movement of a single myosin molecule to be studied.

In one such assay, the sliding-filament assay, the movement of fluorescent-labeled actin filaments along a bed of myosin molecules is observed in a fluorescence microscope. Because the myosin molecules are tethered to a coverslip, they cannot move; thus any force generated by interaction of myosin heads with actin filaments forces the filaments to move along the myosin (Figure 18-22a). If ATP is present, added actin filaments can be seen to glide along the surface of the coverslip; if ATP is absent, no filament movement is observed. Actin filaments always move with the (−) end in the lead. This movement is caused by a myosin head (bound to the coverslip) “walking” toward the (+) end of a filament.

Figure 18-22. The sliding-filament assay.

Figure 18-22

The sliding-filament assay. (a) Schematic diagram illustrates movement of actin filaments across myosin molecules attached to a coverslip. After myosin molecules are adsorbed onto the surface of a glass coverslip, excess myosin is removed; the coverslip (more...)

From video camera recordings of sliding-filament assays, the velocities at which different myosins move an actin filament have been calculated (Figure 18-22b). These rates vary by more than a hundredfold, from 0.04 μm/s for myosin I in the brush border to 4.5 μm/s for myosin II in skeletal muscle. This variation reflects the specific functions of different myosins: Fast movement is associated with muscle contraction, whereas slow movement is adequate for transport in the cytosol.

Researchers have used the sliding-filament assay to identify the role of myosin domains, determine the effects of mutations on myosin, and study how myosin is regulated by other proteins. For example, the two-headed HMM fragment of myosin II, but not the LMM (tail) fragment or single-headed S1 fragment, was found to move actin filaments at velocities comparable with those achieved with intact myosin or myosin filaments. This and other observations pinpoint the myosin head as necessary and sufficient for movement; that is, it is the essential motor domain of the molecule.

Myosin Heads Move in Discrete Steps, Each Coupled to Hydrolysis of One ATP

Given that the myosin head domain is sufficient to cause movement, how much force is generated by a myosin head and how far does it travel when an ATP molecule is hydrolyzed? The forces generated by single myosin molecules can be measured with a device called an optical trap. In this device, the beam of an infrared laser focused by a light microscope on a polystyrene bead (or any other object that does not absorb infrared light) captures and holds the bead in the center of the beam. The strength of the force holding the bead is adjusted by increasing or decreasing the intensity of the laser beam.

If a bead is attached to the end of an actin filament, then an optical trap can capture the filament, via the bead, and hold the filament to the surface of a myosin-coated coverslip (Figure 18-23a). The force exerted by a single myosin molecule on an actin filament is measured from the force needed to hold the bead in the optical trap. A computer-controlled electronic feedback system keeps the bead centered in the trap, and myosin-generated movement of the bead is counteracted by an opposing movement of the trap. The distance traveled by the actin filaments is measured from the displacement of the bead in the trap.

Figure 18-23. Use of the optical trap to determine myosin-generated force.

Figure 18-23

Use of the optical trap to determine myosin-generated force. (a) The light from a single laser beam captures refractive objects — polystyrene beads, bacteria, or cell organelles. (Biological materials do not absorb infrared light (more...)

Such studies show that myosin II moves in discrete steps, approximately 5 – 10 nm long, and generates 1 – 5 piconewtons (pN) of force — the same force as that exerted by gravity on a single bacterium. This force is sufficient to cause myosin thick filaments to slide past actin thin filaments during muscle contraction or to transport a membrane-bounded vesicle through the cytoplasm. With a step size of 5 nm, myosin would bind to every actin subunit on one strand of the filament.

By using an optical trap with a novel imaging method, researchers have recently directly addressed the question of whether hydrolysis of one ATP molecule leads to a one-step movement of myosin (Figure 18-23b). However, the preliminary evidence that hydrolysis and movement are closely coupled is inconclusive; it is not clear whether myosin takes a discrete step for every ATP hydrolyzed.

Myosin and Kinesin Share the Ras Fold with Certain Signaling Proteins

X-ray crystallographic analysis of the myosin S1 fragment has revealed three key pieces of information about the myosin motor domain: its shape, the positions of the essential and regulatory light chains, and the locations of the ATP-and actin-binding sites. The elongated myosin head measures 16.5 × 6.5 × 4.5 nm, and is attached at one end to the α-helical neck (Figure 18-24). Two light-chain molecules lie at the base of the head, wrapped around the neck like C-clamps. In this position, the light chains stiffen the neck region and therefore are able to regulate the activity of the head domain.

Figure 18-24. Three-dimensional structure of a myosin II S1 fragment (see Figure 18-20b).

Figure 18-24

Three-dimensional structure of a myosin II S1 fragment (see Figure 18-20b). X-ray crystallography reveals that the head domain has a curved, elongated shape (16.5 × 6.5 × 4.5 nm) and is bisected by a large (more...)

The surface of the myosin head is marked by a large cleft, extending from the actin-binding site on one side to the ATP-binding pocket on the opposite side. These two crucial binding sites are separated by 3.5 nm, a long distance in a protein. The presence of a surface cleft provides an obvious mechanism for generating large movements of the head domain. We can imagine how opening or closing of a cleft in the head domain, by binding or releasing actin or ATP, causes the head domain to pivot about the neck region. As discussed in detail in the next section, the ATP- and actin-binding sites are most likely coupled by large changes in the conformation of the head domain.

The core of the myosin motor domain is structurally similar to that of kinesin, a microtubule motor protein. Both motor domains contain the so-called Ras fold, in which a nucleotide molecule is bound to loops at one end of a β-sheet domain. The loops, called P-loops, are characteristic of ATPase active sites. This structural motif was initially identified in Ras protein, a GTP-binding protein that functions in certain intracellular signaling pathways (see Figure 3-5). The presence of the Ras fold in myosin and kinesin suggests that these motor proteins may have evolved from ancient nucleotide-binding proteins. The differences between the structures of myosin and kinesin reveal how they bind to actin and microtubules, respectively, and how binding is coupled to ATP hydrolysis.

Conformational Changes in the Myosin Head Couple ATP Hydrolysis to Movement

Knowing the three-dimensional structure of the myosin head and the kinetics of the myosin ATPase, researchers could begin to understand how myosin harnesses the energy released by ATP hydrolysis to generate the force for movement. In the following discussion, we will consider the general case in which a myosin molecule walks along an actin filament. Because all myosins are thought to move using the same mechanism, we will ignore for the moment whether the myosin is bound to a vesicle or is part of a thick filament (as in muscle). One assumption in the model we describe is that the hydrolysis of a single ATP molecule is coupled to each step taken by a myosin molecule along an actin filament. As noted previously, some evidence indicates that this is true.

As shown in Figure 18-25, myosin undergoes a series of events during each step of movement. Repetition of this cycle causes myosin to slide relative to an actin filament. During one cycle, myosin must exist in at least three conformational states: a prehydrolysis ATP state unbound to actin, an ADP-Pi state bound to actin, and a state after the power stroke is completed. The major question to answer is how the nucleotide-binding pocket and the distant actin-binding site are mutually influenced and how changes at these sites are converted into force. Structural studies implicate the cleft as the physical link that communicates by domain movements. In structures of myosin bound to nucleotide analogs that mimic the prehydrolysis state and the transition state for hydrolysis, the presence of the γ-phosphate group of ATP and binding to actin control whether the cleft is open or closed. Binding of ATP to myosin opens the cleft, causing a disruption at the actin-binding site at the opposite end of the cleft. After ATP hydrolysis, the cleft closes partially. This conformational change traps the hydrolysis products and restores the actin-binding site. In addition, large rotations near the neck probably prepare myosin for the power stroke. An integral part of this model is that conformational changes in the head and neck are transmitted and amplified to other parts of the molecule through the light chains bound to the neck.

Figure 18-25. The coupling of ATP hydrolysis to movement of myosin along an actin filament.

Figure 18-25

The coupling of ATP hydrolysis to movement of myosin along an actin filament. In the absence of bound nucleotide, a myosin head binds actin tightly in a “rigor” state. When ATP binds (step 1 ), it opens the cleft in the head, disrupting (more...)

In the model depicted in Figure 18-25, myosin slides along an actin filament; however, the type of movement that occurs depends on how myosin or actin is anchored. For example, in a bipolar thick filament, the myosin II heads are firmly anchored to the thick filament backbone (see Figure 18-21b). Because the myosin heads in the two halves of a thick filament have opposite polarities, actin filaments slide toward the middle of the thick filament while the thick filament remains immobile. In contrast, a single myosin I molecule, bound to a membrane-bounded vesicle, moves along an actin filament because the actin filament is part of a massive structure, the cytoskeleton. Thus the frame of reference for movement changes depending on whether actin or myosin is immobile.


  •  Myosins are motor proteins that interact with actin filaments and couple hydrolysis of ATP to conformational changes that result in the movement of myosin and an actin filament relative to each other.
  •  Genomic analysis has revealed 13 different myosins. All consist of a highly conserved head (motor) domain, which is an actin-activated ATPase responsible for generating movement; a neck domain, which is associated with several regulatory light-chain subunits; and an effector tail domain, which is unique to each type of myosin and determines its specific functions in cells (see Figure 18-20).
  •  Myosin II, a dimeric molecule with a long rodlike tail domain, assembles into bipolar thick filaments that take part in muscle contraction.
  •  In the presence of ATP, the dimeric head domain of myosin II alone can generate movement.
  •  The actin-binding site and ATP-binding pocket in the head domain of a myosin molecule are distant from each other but connected by a surface cleft. This cleft is thought to be critical in coupling ATP hydrolysis to myosin movement.
  •  Movement of myosin results from attachment of the myosin head to an actin filament, bending of the head, and its subsequent detachment in a cyclical ATP-dependent process (see Figure 18-25). During each cycle, myosin moves 5 – 25 nm and one ATP is hydrolyzed.
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Copyright © 2000, W. H. Freeman and Company.
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