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Myosin Biochemistry of Metabolism: Cell Biology

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Presentation on theme: "Myosin Biochemistry of Metabolism: Cell Biology"— Presentation transcript:

1 Myosin Biochemistry of Metabolism: Cell Biology
Copyright © by Joyce J. Diwan. All rights reserved.

2 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 a diagram accessible from the Myosin Home Page .

3 Myosin II was first studied for its role in muscle contraction, but it functions also in non-muscle cells. Myosin II includes 2 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.

4 2 Light chains, designated essential & 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. The binding site for each light chain is an IQ (isoleucine, glutamine) sequence motif: IQxxxRGxxxR. The 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++.

5 Myosin I has only one heavy chain with a single motor domain.
Its relatively short tail lacks the heptad repeats that would be involved in dimerization via formation of a coiled coil. Myosin VI tail includes a short segment of heptad repeats. Myosin VI is found to be either monomeric or dimeric under different conditions.

6 Myosin V has 2 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 cargo-binding domain at the end of each heavy chain tail.

7 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.

8 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. Movie & amination of actin filament movement driven by immobilized myosin (University of Vermont website)

9 Myosin II heads interact with actin filaments in a reaction cycle that may be summarized as follows:
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. Pi 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. See an animation by the Vale Lab.

10 ATP binds to the myosin head adjacent to a 7-stranded b-sheet.
Loops extending from b-strands interact with the adenine nucleotide.

11 The nucleotide-binding pocket is opposite a deep cleft that bisects the actin-binding domain of myosin. Opening & 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.

12 Similarities in structure of the ADP/ATP-binding site in myosin & the nucleotide binding site in the family of small GTP-binding proteins such as Ras, suggest that myosin may be distantly related to the GTP-binding proteins. There is little sequence homology, but the structural similarity suggests a common ancestor.

13 Consistent with the predicted conformational cycle, different conformations of myosin head & neck are found in crystal structures. b-sheet adjacent to nucleotide-binding site is magenta; light chain backbones green, red.

14 Bipolar complexes of myosin II form by interaction of antiparallel coiled coil tail domains.
These may contain many myosin molecules, as in thick filaments of skeletal muscle. 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 plus ends of adjacent actin filaments.

15 Muscle sarcomere structure, and the role of myosin II in muscle contraction, will not be discussed here. In non-muscle cells, myosin II (the type in muscle) is often found to be associated with actin filament bundles. Existence of bipolar myosin assemblies has been postulated. Contraction is postulated to involve myosin-mediated sliding of antiparallel actin filaments.

16 Contraction is postulated to involve myosin-mediated sliding of antiparallel actin filaments, e.g., in: stress fibers, bundles of actin filaments that link to the plasma membrane where cells attach to the extracellular matrix. belts of actin filaments that encircle epithelial cells, associated with adhering junctions. the contractile ring of cytokinesis, located just inside the plasma membrane at the division furrow. the cortical web of actin filaments, located just inside the plasma membrane in many cells.

17 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 motor domains contact distal tail domains preventing formation of bipolar complexes. 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.

18 Myosin V (diagram in website of X
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.

19 Regulation by Ca++ varies, depending on the type of myosin, the tissue & the organism. E.g.:
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.  Caldesmon, a protein regulated by phosphorylation and by Ca++,  controls actin-myosin interaction in smooth muscle.

20 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.

21 Within microvilli of intestinal epithelial cells, myosin I may have a role in pulling the plasma membrane along actin filament bundles within the microvilli, as they grow by addition of actin monomers at the tip. 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. Myosin VI, which is unique among myosins in walking along actin filaments toward the minus end, has a role in clathrin-mediated endocytosis, as endocytic vesicles are transported inward, away from the plasma membrane.

22 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. The processive movement of myosin V is appropriate for its role in transporting organelles along actin filaments.

23 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.

24 Myosin V step length has been measured by monitoring movement of individual fluorescent labeled calmodulin light chains associated with the myosin V neck domain. Diagrams in article by Yildiz et al. & U. Illinois website. High resolution EM has detected conformations also consistent with the hand-over-hand stepping mechanism.

25 Animation: Myosin V walking along an actin filament.
High resolution electron microscopy has detected conformations consistent with the hand-over-hand stepping mechanism. Animation: Myosin V walking along an actin filament. Based on electron microscopic images of myosin V fragments (part of the tail domain with 2 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.)

26 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 (+) ends 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.

27 For example: 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 & 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 & highly branched.

28 Myosin I, which binds to the plasma membrane, may pull the membrane forward as it walks along actin filaments toward the plus end. 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.

29 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. 

30 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: Website with movies (FishScope website) Website with movies, animation & diagram. (IMB Salzburg)

31 Signaling in ameboid movement is complex. Examples:
Regulatory roles of members of the Rho family of GTP-binding proteins include: Rac-GTP activates Scar/Wave (WASP family), 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 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.

32 PIP2 hydrolysis by signal-activated Phospholipase C may result in localized increases in profilin, cofilin, gelsolin, and Ca++ (due to IP3 release). 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.

33 Summary of roles of some cell constituents in ameboid movement.

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