Actin and Myosin


The skeleton of a cell is referred to as the cytoskeleton. The cytoskeleton accounts for the majority of a cell’s mass and provides the cell with rigidity and shape, much like the skeletal system of the body. However, unlike bones of the animal skeletal system, the components of the cytoskeleton are ever-changing in order to drive dynamic processes such as cellular movement and cellular division.

Adding to the dynamic nature of the cytoskeleton are various motor proteins that bind to and move along cytoskeletal components. This exercise will focus on the motor protein myosin, which associates with actin filaments in order to do work within a cell. An actin and myosin pair found in skeletal muscle, for example, does the work necessary to drive muscle contraction.

The cytoskeletal protein, actin, the mechanism of muscle contraction, and the myosin reaction pathway are featured in detail over the course of this exercise.

Learning objectives:


The cytoskeleton refers to the structural components of a cell. Microfilaments, microtubules, and intermediate filaments are the three predominant components comprising the cytoskeleton and each is a polymer of assembled protein subunits. Collectively, these three cytoskeletal components determine the physical attributes of a cell.

Actin and tubulin are round and compact globular protein subunits that assemble to form microfilaments and microtubules, respectively. In contrast with microfilaments and microtubules, intermediate filaments are made up of long, fibrous protein subunits.

Actin, the building block of microfilaments, will be discussed in greater detail over the course of this exercise.


A microfilament is composed of repeated subunits of the globular protein called actin. Notice how the protein consists of two domains separated by a cleft. Actin is able to bind the adenine nucleotides ATP or ADP within its nucleotide binding cleft.


A microtubule is composed of repeated subunits of the tubulin heterodimer. The two different subunits that comprise the heterodimer are called a-tubulin and b-tubulin. Tubulin subunits bind guanine nucleotides.

Intermediate filaments

Various fibrous proteins assemble to form different types of intermediate filaments. One example of an intermediate filament is made up of repeated subunits of the protein keratin. Keratins do not bind a nucleotide.


Actin assembles to form long protein polymers referred to as microfilaments. Polymerized actin is filamentous, and is sometimes referred to as F-actin to distinguish it from its globular monomeric form, G-actin. Actin subunits assemble with their nucleotide binding clefts oriented in the same direction and the resulting filament looks like two chains that are loosely wrapped around one another.

After an actin subunit containing ATP is added to a growing microfilament, ATP is hydrolyzed to ADP plus inorganic phosphate. Upon release of inorganic phosphate, a conformational change takes place, switching actin from an open, ATP-bound state to a closed, ADP-bound state.

Similar to the two ends of a magnet, the ends of a microfilament are distinguishable from one another, and have defined polarity. The end of the microfilament closest to relatively exposed nucleotide binding clefts is referred to as the minus end. The other end, where clefts are buried, is the plus end. Typically, the rate of polymerization is greater at the plus end than at the minus end.


Because microfilaments can be assembled, remodeled, and disassembled, they are considered to be dynamic structures. Dynamic structures allow a cell to change shape, facilitating cellular movement. White blood cells, for example, remodel microfilaments in order to cross capillary walls and enter damaged tissue, en route to participating in wound healing.

One dynamic process of microfilaments is referred to as treadmilling. Treadmilling occurs when the net rate of addition of actin subunits to one end of a microfilament equals the net rate of removal of actin subunits from the other end.

Various proteins can regulate microfilament dynamics. Capping, branching, and severing proteins are examples of microfilament regulatory proteins that cells employ in order to change shape.


Microfilaments can also be used to generate tensile force when partnered with the motor protein, myosin. Myosin represents a family of proteins involved in myriad processes. In higher organisms, one of these processes is muscle contraction, which is carried out by muscle myosin.

Like all known motor proteins, myosin moves along a cytoskeletal substrate by converting chemical energy into mechanical energy. Myosin’s cytoskeletal substrate is the actin subunit of a microfilament, and its source of chemical energy is ATP. A series of small conformational changes that are tightly linked to nucleotide-associated events occurs within the protein. The end result is a large conformational change referred to as the power stroke. Ultimately, the net result is the generation of tensile force within a cell.


In order to understand muscle contraction, let us first review the anatomy of a muscle.


Muscle myosin exists in the cell as a hexamer, containing two identical heavy chains and two pairs of different light chains. The myosin heavy chain consists of three major domains: the head, neck, and tail. One half of the heavy chain folds into the globular head domain, which contains the binding sites for actin and an adenine nucleotide. These two binding sites define the head domain as the catalytic portion of the protein. The long, fibrous tail domain formed from the other half of the myosin heavy chain assumes an alpha helical conformation.

Two tail domains twist around each other to form a single rodlike structure, thereby dimerizing two myosin heavy chains.

Myosin’s helical neck domain connects the head and tail domains. A pair of light chains associates with the myosin neck domain and helps to stiffen the neck helix so that it can act as an effective lever. The lever acts to move the myosin head in a large sweeping step called the power stroke, causing muscle contraction.


The chemical energy derived from ATP hydrolysis is used to fuel myosin’s mechanical activity, much like gasoline is used to fuel a car’s engine in order to move. This motor protein’s movement along an actin filament is tightly linked to the events associated with ATP hydrolysis. Therefore, the events at the nucleotide binding site must be linked to the other regions of the protein that generate movement. Because a distance of approximately 35 Å separates the nucleotide binding site from the actin binding site, a series of small conformational changes is responsible for transmitting information between the various domains.

Imagine trying to talk to a friend from a distance using two tin cans. Unless a wire connects the two cans, the voice of one person is inaudible to the other person. Myosin’s nucleotide binding site and actin binding site are analogous to the tin cans. In order to transmit information between these catalytic domains, structural motifs must connect the different domains like a wire connecting two tin can telephones.

Two moieties within myosin are essential in communicating nucleotide events to distant movement-generating domains. The two domains are called the gamma phosphate sensor and the relay helix. The gamma phosphate sensor is a protein segment positioned very close to the nucleotide binding site that allows myosin to distinguish between ATP- and ADP-bound states. The relay helix, in turn, is a long helical structure that transmits information from the gamma phosphate sensor to other regions of the protein, including the actin binding site and lever arm.

The series of small conformational changes ultimately adds up to a large conformational change, as myosin's helical lever arm is rotated approximately 70°, causing the myosin head to move 50-100 Å to the middle of the sarcomere. This large conformational change is called the power stroke, and is the force-generating step that results in muscle contraction.


The movement of myosin along an actin filament is described as a “hopping” motion. This is because the actin subunit is released by myosin following each round of the reaction cycle, instead of remaining bound to the actin filament while covering a large distance.

  1. ATP binding alters the configuration of the myosin head so that it releases actin.
  2. Rapid hydrolysis of ATP to ADP plus inorganic phosphate occurs. A conformational change rotates the lever arm so that the myosin head is cocked away from the sarcomere’s mid-line.
  3. The hydrolysis of ATP results in the myosin head binding an actin subunit farther along the thin filament.
  4. Inorganic phosphate and ADP are released. As these reaction products exit, the myosin lever arm rotates in a step commonly referred to as the power stroke, which results in cocking the myosin head towards the sarcomere’s mid-line. Following ADP release, the myosin head is ready to bind ATP and start the reaction cycle anew.