Biochemistry of Metabolism: Cell Biology

Myosin

Contents of this page:
Classes of myosin & basic structure
Motor domain function & structure
Bipolar assemblies of myosin II
Regulation
Roles & mechanisms of myosins I, V, & VI
Ameboid movement

Note: In these notes references are given to page numbers in the Molecular Biology of the Cell textbook by Alberts et al. (A).

Myosins  are a large superfamily of motor proteins that move along actin filaments, while hydrolyzing ATP. About 20 classes of myosin have been distinguished on the basis of the sequence of amino acids in their ATP-hydrolyzing motor domains. The different classes of myosin also differ in structure of their tail domains. Tail domains have various functions in different myosin classes, including dimerization and other protein-protein interactions. Only a few of the known classes of myosin will be discussed here.
See diagrams in A. p. 950, 951, a diagram accessible from the Myosin Home Page at Cambridge University, and diagrams depicting the motor domain and neck region below.

Myosin II was first studied for its role in muscle contraction, but it functions also in non-muscle cells. Myosin II includes two heavy chains. The globular motor domain of each heavy chain catalyzes ATP hydrolysis, and interacts with actin. Each heavy chain continues into a tail domain in which heptad repeat sequences promote dimerization by interacting to form a rod-like a-helical coiled coil.

Two light chains, designated essential and regulatory, wrap around the neck region of each myosin II heavy chain. In addition to regulatory roles, light chains may help to stiffen the neck regions. The binding site for each light chain is an IQ (isoleucine, glutamine) sequence motif: IQxxxRGxxxR. Myosin II light chains are similar in structure to calmodulin, but in many organisms have lost the ability to bind Ca++. However, the calmodulin-like light chains of some myosins do bind Ca++.

Myosin I has only one heavy chain with a single globular motor domain. Its relatively short tail lacks the heptad repeats that would be involved in dimerization via formation of a coiled coil.

The Myosin VI tail domain includes a short segment of heptad repeats. Myosin VI is found to be either monomeric or dimeric under different conditions.

Myosin V has two heavy chains like myosin II. But myosin V has a longer neck region that has 6 binding sites for calmodulin light chains. Its shorter coiled coil region is followed by a globular domain at the end of each heavy chain tail.

Motor domains of most myosins move along actin filaments toward the plus ends of the filaments. This movement is ATP-dependent and is accompanied by ATP hydrolysis. 

An exception is myosin VI, which moves toward the minus ends of actin filaments.

Proof that the head domain with attached neck is sufficient to drive movement has been obtained in studies of isolated myosin heads, using fluorescence microscopy. Myosin heads, detached from myosin tails by protease treatment and fixed to a glass surface, promote gliding of actin filaments labeled with fluorescent rhodamine-phalloidin. This movement is ATP-dependent. See Alberts et al. p. 951.

See also a movie and animation of actin filament movement driven by immobilized myosin, in University of Vermont website.

Myosin II heads interact with actin filaments in a reaction cycle that may be summarized as follows (diagram  in A p. 955):

• ATP binding causes a conformational change that causes myosin to let go of actin.
• The active site closes, and ATP is hydrolyzed, as a conformational change (cocking of the head) results in  myosin weakly binding actin, at a different place on the filament.
• P_i release results in conformational change that leads to stronger myosin binding, and the power stroke.
• ADP dissociation leaves the myosin head tightly bound to actin. In the absence of ATP, this state results in muscle rigidity called rigor.

An animation may be viewed at a website of the Vale Lab at University of California, San Francisco.
 

ATP binds to the myosin head adjacent to a 7-stranded b-sheet. Loops extending from b-strands interact with the adenine nucleotide.  The nucleotide-binding pocket of myosin is opposite a deep cleft that bisects the actin-binding domain (diagram in A p. 953). Opening and closing of the cleft is proposed to cause the head to pivot about the neck region, as occupancy of the nucleotide-binding site changes and as myosin interacts with and dissociates from actin.

Consistent with the predicted conformational cycle, different conformations of the myosin head & neck have been found in crystal structures. Two examples are shown. The b-sheet adjacent to the nucleotide-binding site is colored magenta; light chains are displayed as backbone, in green & red.

Bipolar complexes of myosin II form by interaction of antiparallel coiled coil tail domains. These complexes may contain many myosin molecules, as in the thick filaments of skeletal muscle (diagram in A p. 950). Antiparallel actin filaments may be caused to move relative to one another, as motor domains at the opposite ends of bipolar myosin II complexes walk toward the plus ends of adjacent actin filaments. Muscle sarcomere structure and the role of myosin II in muscle contraction will not be discussed in detail here, since it is covered in other courses at Rensselaer. (If you are not familiar with the role of myosin II in muscle see A p. 961-964.)

In non-muscle cells, myosin II (the type in muscle sarcomeres) is often found to be associated with actin filament bundles. Existence of bipolar myosin assemblies has been postulated. Contraction of actin filament bundles is postulated to involve myosin-mediated sliding of antiparallel actin filaments, e.g., in each of the following:

• stress fibers, bundles of actin filaments that link to the plasma membrane at sites where a cell attaches to the extracellular matrix (A p. 940).

• belts of actin filaments that encircle epithelial cells, associated with adhering junctions (A p. 1071-1072).

• the contractile ring of cytokinesis, located just inside the plasma membrane at the division furrow (A p. 1054).

• the cortical web of actin filaments, located just inside the plasma membrane in many cells.

Regulation by phosphorylation:

• Myosin II of smooth muscle as well as non-muscle cells may be regulated by phosphorylation of its regulatory light chains.
  • Dephosphorylation stabilizes an inhibited bent conformation in which the motor domains contact distal tail domains preventing formation of bipolar complexes. Diagram in A p. 961.
  • Phosphorylation catalyzed by Myosin Light Chain Kinase or Rho Kinase activates by promoting transition to the extended conformation.
• Myosin II in Dictyostelium transitions to an inhibited bent conformation unable to form bipolar filaments when residues of its tail domain are phosphorylated via a Myosin Heavy chain Kinase.
• Myosin V (diagram in website of X. Li) and the microtubule motor protein kinesin are also inhibited by regulated transition to bent conformations. In the bent conformation of each of these motor proteins, interaction of a globular tail domain with the motor domain inhibits its ATPase activity. Binding to a cargo protein for which it has affinity promotes transition to an active, non-bent state.

Regulation by Ca⁺⁺ varies, depending on the type of myosin, the tissue and the organism. For example:

• Some myosins are regulated by binding of Ca⁺⁺ to calmodulin-like light chains, in the neck region.
• A complex of tropomyosin and troponin (which includes a calmodulin-like protein) regulates actin-myosin interaction in skeletal muscle sarcomeres. (See A p. 965)
• Caldesmon, a protein regulated by phosphorylation and by Ca⁺⁺,  controls actin-myosin interaction in smooth muscle.

Myosins I, V, & VI bind to membranes or to macromolecular complexes via globular tail domains. They have roles, e.g., in movements of organelles or plasma membranes relative to actin filaments:

• Myosins I & V associate with Golgi membranes and with vesicles derived from the Golgi, including synaptic vesicles.  In mice, myosin V mutations lead to defects in synaptic transmission.
• In skin melanocytes, myosin V is involved in movement of membrane-enclosed pigment granules into dendritic cell extensions (A p. 959).
• Within microvilli of intestinal epithelial cells, myosin I may have a role in pulling the plasma membrane along actin filaments bundles within the microvilli, as they grow by addition of actin monomers at the tip (A p. 942).
• A member of the myosin I class of motor proteins (myosin Ic) has a special role in hearing, relating to movement of membrane-embedded ion channels along the surface of stereocilia, thin cell processes that contain actin filaments (A p. 1270).
• Myosin VI, which is unique among myosins in walking along actin filaments toward the minus end, has a role in clathrin-mediated endocytosis (A p. 752) as endocytic vesicles are transported inward, away from the plasma membrane.

Movement of myosin V along actin is processive, meaning that myosin V remains attached to an actin filament as it walks along that filament. In contrast, myosin II is a non-processive motor that detaches from actin at a stage of each reaction cycle (see above). The processive movement of myosin V is appropriate for its role in transporting organelles along actin filaments. In the hand over hand stepping mechanism of myosin V, one head domain dissociates from an actin filament only when the other head domain binds to the next subunit with the correct orientation along the helical actin filament. Since there are 13 actin subunits per helical turn, myosin V has a relatively long step length of 74 nm. By stepping the length of the actin helical repeat, myosin V maintains a straight path along an actin filament, rather than spiraling around it.

Myosin V step length has been measured by monitoring movement of individual fluorescent labeled calmodulin light chains associated with the myosin V neck domain.  For diagrams, see article by Yildiz et al. and a University of Illinois website on research of P. Selvin.

High resolution electron microscopy has detected conformations consistent with the hand-over-hand stepping mechanism.

Animation: This animation of myosin V walking along an actin filament is based on electron microscopic images of myosin V fragments, consisting of part of the tail domain with two attached heads, attached to actin filaments in what is interpreted as different stages of the reaction cycle.  (By M. L. Walker, S. A. Burgess, J. R. Sellers, F. Wang, J. A. Hammer, J. Trinick & P. J. Knight.)

Ameboid movement: At the leading edge of a moving cell is the lamellipodium. Forward extension of a lamellipodium is driven by actin polymerization. Lamellipodia contain an extensively branched network of actin filaments, with their plus ends oriented toward the plasma membrane. Localization of proteins that participate in generating forward movement, at the leading edge or other regions of an advancing cell, has been demonstrated, e.g., by fluorescent labeling. See A p. 974-977. Some examples discussed above and in the notes on actin:

• Profilin promotes ADP/ATP exchange by G-actin, to yield the ATP-bound form competent to polymerize, at the leading edge of an advancing cell. 

• Arp2/3, a complex that includes actin related proteins 2 & 3, binds to the sides of actin filaments and nucleates growth of new filaments within lamellipodia.

• Capping protein adds to the plus ends of actin filaments shortly after they are nucleated by Arp2/3, keeping actin filaments at the leading edge short and highly branched.

• Myosin I binds to the plasma membrane, and may pull the membrane forward as it walks actin filaments toward the plus end (diagram above). 

• Cofilin and gelsolin may sever actin filaments, providing new plus ends for nucleation of actin filament growth and helping to keep actin filament branches short within the lamellipodium.
  Cofilin also promotes depolymerization of actin filaments further back from the leading edge within a lamellipodium.

Various cross-linking proteins stabilize the actin network in lamellipodia. Pulse labeling has shown that the newly formed actin filaments are stable, as an advancing lamellipodium moves past them, until they disassemble further back from the edge.  Myosin II is located predominantly at the rear end of a moving cell, or in regions being retracted. Contraction in these regions probably involves sliding of antiparallel actin filaments driven by bipolar myosin assemblies. When a focal adhesion fails to detach, a fragment of cytoplasm is sometimes left behind. Calpains (intracellular Ca++-activated proteases) may degrade constituents of focal adhesions at the rear of a moving cell as it is pulled forward. See FishScope website with movies. See a website of the Institute of Molecular Biology at Salzburg with movies, an animation & a diagram.

Signaling in ameboid movement is complex and only a few aspects of this regulation will be summarized here. For example:

Regulatory roles of members of the Rho family of GTP-binding proteins include:

• Rac-GTP activates Scar/Wave (a member of the WASP family of proteins), which in turn activates Arp2/3 to nucleate formation of actin filament branches at the leading edge of a moving cell.
• Rho-GTP activates Rho Kinase (ROCK) to phosphorylate myosin II regulatory light chains, to promote interaction of myosin II with actin filaments. This is essential for formation of stress fibers and contraction of these stress fibers at the rear of a moving cell.
  Rho-GTP also activates formins to promote formation of the linear actin filaments found in stress fibers.

Ca⁺⁺ indicator dyes have been used to show that cytosolic [Ca⁺⁺] is highest at the rear of an advancing cell, where it may activate Myosin Light Chain Kinase and calpains. Cytosolic [Ca⁺⁺] is relatively low at the leading edge of an advancing cell, where movement is driven more by actin filament assembly.

A summary of roles of some cell constituents in ameboid movement is presented at right. See also diagrams by Vicente-Manzanares et al. in J. Cell Science.

For more details, see the Myosin Home Page, which provides links to additional sites with information relating to myosin.

Copyright © 1998-2007 by Joyce J. Diwan. All rights reserved.

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