| Myo I, II and V | The nature of the myosin tail determines what it binds to and therefore what it does. Myo I binds membranes and is involved in endocytosis. Myo II forms dimers that associate to each other in a bidirectionally symmetrical configuration to give rise to a myosin II bouquet. Myo V dimerises but does not form large complexes - but binds via adaptor molecules to vesicles (for organelle transport/positioning), microtubules (to direct cell polarity) and RNA (for cell differentiation) |
| Myosin II filaments | Myosin II filaments are formed from bi-symmetrical bouquets of Myosin II dimers. Each dimer is tied together by a coiled-coil tail region which can mostly be removed by Chymotrypsin cleavage but the heads remain together. Further protease digestion with Papain removes the remainder of the tail (called the S2 region) leaving monomeric S1 heads which have the motor activity and both essential and regulatory light chain binding sites. |
| Arrowheads | The polarity gives actin filaments a repeated arrowhead appearance with the arrow pointing to the minus end of the filament. |
| Myosin motility assay | Myosin motility can be studied by binding myosin to a coverslip, then watching fluorescent actin filaments moving over it. This assay and structural studies have been useful in delineating the molecular mechanism of myosin action |
| Myosin motility steps | 1. ATP binds to S1 heads, and hea is released from actin. 2. The ATP molecule gets hydrolysed to ADP + Pi and the myosin head rotates into "cocked" state. 3. Myosin head binds actin filament. 4. "Power Stroke": Release of P and elastic energy straightens myosin; moves actin filament left. 5. ADP released and ATP is bound, head released tec. |
| Dissociation of Pi | Actin absence: The dissociation of Pi is very slow. Actin addition: raises the rate of Pi dissociation 300 fold
to approximately 10 ATP molecules per myosin per sec . The dissociation of Pi also induces the “power stroke” for myosin action. The release of Pi causes small conformational changes in the head (motor) domain. This is amplified by a “converter” region at the base of the head which acts as a fulcrum to cause the leverlike neck to rotate. This in turn is amplified by the rodlike lever arm, which constitutes the neck domain, so the actin filament moves by a few nanometers. |
| Myosin cycle | Myosin is tightly bound to actin filaments without nucleotide (rigor) and releases from the actin filament when ATP binds, cocking is induced by ATP hydrolysis and the power stroke is provided by dissociation of Pi |
| Kinesin cycle | Kinesin is bound to MTs (in rigor) with ATP bound and is released when ATP is hydrolysed to ADP and ATP binding produces the power stroke that throws the partner head forward. |
| Muscle cells | Muscle cells are syncytial (multi-nucleated) up to ~50cm in length formed by the fusion of myoblasts. Muscle cells contain a number of myofibrils each of which consists of a regular repeating array of sarcomeres, the base unit of muscle action. Electron micrographs show alternating light and dark bands with a line (the Z line) down the middle of a light band. Each sarcomere is defined as the region between the Z lines and has one dark band and one half of a light band on either side. The dark band are
composed of myosin II filaments and the light band of actin filaments. The darkest region is the region of overlap containing both myosin filaments and actin filaments arranged in a regular pattern. When the muscle contracts the light band disappears as the region of overlap between the myosin and actin filaments increases |
| CapZ, Tropomodulin, Nebulin and Titin | Each myosin thick filament is surrounded by 6 thin microfilaments (actin filaments) for it to work on. The plus ends of the actin filaments are capped by CapZ which allows them to be buried in the Z disk. The minus ends of the actin filaments are capped by tropomodulin. Nebulin is a large molecule that has repeated actin binding sites and is thought to determine the length of the actin filaments. Titin is one of the biggest proteins in the human body and is an elastic molecule that prevents over-stretching of the sacromere. When you pull a muscle you rip the titin protein apart. |
| Triggering contraction | Nerve impulses trigger muscle contraction by causing depolarisation of the plasmamembrane (sarcolemma). Key to this are Transverse tubules (T-tubules) which are invaginations of the plasmamembrane which lie adjacent to the outer face of the sarcoplasmic reticulum (SR) around each myofibril. An action potential causes opening of a voltage gated Ca2+ channel which release a small burst of Ca2+ into the cytosol. This Ca2+ binds to a channel in the SR which triggers massive and explosive release of Ca2+ into the cytosol. The increase in calcium concentration causes myosin to bind to actin. |
| Ca2+ and conformational changes | Elevated Ca2+ concentration causes a conformational change in two accessory proteins, tropomyosin (TM) and the troponin (TN) complex. In the absence of Ca2+ tropomyosin, which is a rope like protein, covers up the binding site for myosin on the actin filament. In the presence of Ca2+ the myosin binding site is exposed. Troponin complex is composed of 3 molecules, TH-T, TN-I and TN-C. TN-C is the calcium binding subunit of the complex. Association of Ca2+ to TN-C causes a conformational change which ultimately results in a conformational change in tropomyosin. |
| Cardiac muscle | Cardiac muscle is striated not syncytial. Gap junctions enable the action potential to spread rapidly from cell to cell so that the actions of all muscle cells are co-ordinated resulting in a rhythmic heart beat. The filament genes (actin, myosin etc.) are different from skeletal muscle, but otherwise the sarcomere functions in much the same way. |
| Cyclin B / Cdk1 kinase | Formation and contraction of the cytokinetic actomyosin ring is dependent on signals from the cell cycle machinery. During mitosis Cyclin B / CDK1 phosphorylates myosin light chain (MLC) and this prevents Myosin II from functioning. When CDK1 is inactivated at anaphase dephosphorylation of MLC allows myosin light chain kinase (MLCK) to phosphorylate MLC at Ser19. This allows myosin bundle formation at the cleavage furrow and constriction of the actomyosin ring. |
| Laser based optical tweezers | The step size of a myosin motor can be calculate by laser based optical tweezers. In this approach the myosin is bound at low density to an immobilised bead. An actin filament, which is held by two laser based light sources, is lowered towards the bead. When myosin touches the actin filament and ATP is added the ATPase cycle is stimulated and myosin moves the actin filament. The distance and force by which it does this can be calculated via the computer running the microscope. |
| The model predicts | That the distance a particular myosin motor moves must be proportional to the length of the neck region. To test this the velocity by which mutant version of a myosin which have shorter neck regions was measured. This showed a remarkable correlation between neck length and velocity which is due to a change in step size. |
| Myosin II | It takes approximately 5-10 nm steps and then releases from the actin filament so the distance moved returns to zero as there is no force applied during this time. Myo II only spends 10% of its time attached to an actin filament (Duty ratio = 10%). Because myosin II filaments in muscle have multiple single heads -one or more is always bound otherwise muscle wouldn’t be able to do work. |
| Myosin V | By contrast Myosin V takes successive 36nm steps as so it described as processive. The duty ratio = 70%, so one of its two heads are always bound to the actin filament |
| How does Myosin V travel (hand over hand) | Researchers managed to attach a fluorescent probe to only one of the two myosin heads. This revealed that Myosin V moves by taking 72nm steps. Indeed this is almost exactly the distance between the successive helical turns on the actin filament so Myosin V walks down only one side of the actin filament. This is precisely the properties you would expect for a good processive motor. |
| Myosin V uses | Myosin V can have multiple functions in a single cell. For example in budding yeast Myosin V through binding various adaptor proteins can transport to the growing bud membrane cargoes, microtubules and specific mRNAs. Myosin V mediates the asymmetric segregation of the ash1 mRNA in budding yeast. Ash1p protein ensures that only the mother cell changes mating type. ash1 mRNA is tethered to Myosin V through adaptor proteins, She2 and She3. Actin filaments are nucleated at the bud tip by formin. Myosin V travels towards the the plus end of actin filaments bringing ash1 mRNA with it. ash1 mRNA is only translated in the bud cell to prevent differentiation. |
| Chemotaxis | Chemotaxis is the ability to sense and move towards or away from a directional signal. This is important for multiple functions in the human body including directed cell movements during development, the inflammatory response to injury and wound healing |
| Actin binding toxins and the residues required for their binding on the actin monomer | Cytochalasin D: Binds monomers and barbed ends, inhibits polymerization.
Tolytoxin: Binds monomers and barbed ends, inhibits polymerization 1Kx more effective.
Latrunculin A: Binds monomers and inhibits polymerization.
Phalloidin: Binds and stabilizes F-actin, can be labelled with a fluorescent dye, making it very useful for staining actin filaments in cells. |
| Locomotion steps | 1) Locomotion begins with the extension of one or more Lamellipodia from the leading edge of the cell. This involves actin polymerisation which pushes the membrane forward. 2) New focal adhesions are
established which involves capping of actin filament bundles at specialised sites on the plasma-membrane 3) The bulk of the cytoplasm is pushed forward by contraction of Actin-Myosin II bundles (stress fibres) at the rear of the cell in a process called Translocation. 4)The trailing edge of the cell detaches from the Extracellular matrix and retracts into the cell body. During this process the endocytic machinery internalises Integrins and transports them to the front of the cell to be used again. |
| Components | Stress fiber: contractile bundle, Actin and Myo II
cell cortex: gel-like network, Filamin
filopodium: tight parallel bundle, Fimbrin
lamellipodia: branched network, Arp2/3 complex |
| Steps during chemotaxis | During chemotaxis the intracellular distribution of each class of GTPase is controlled so that a gradient is formed within the cell. At the leading (growing) edge Cdc42 activation promotes formin and Arp2/Arp3 dependent actin assembly to promote filopodia and lamellipodia growth. This in turn stimulates Rac GTPase which further promotes branched actin network assembly behind the leading edge. Rac activation leads to Rho GTPase activation at the lagging edge of the cell which promotes stress fiber formation and myosin II activation which powers forward movement of the bulk of the cell contents. This systems is highly dynamic as the cell can rapidly change direction in response to changes in concentration and direction of chemotactic signals. |
| Regulation of actin assembly by Rho family GTPases | Microinjection of dominant activated forms of the Cdc42 (membrane spikes, Filapodia), Rac (membrane ruffles, Lamellipodia) and Rho small GTPases (stress fibers) have different effects on actin filament organization in cells |
| An assay for wound healing in vitro | Chemotaxis is important for multiple responses in the human body including wound healing and for inflammatory response. In this in vitro wound healing assay a confluent layer of cells in a petri dish are disturbed. Cells migrate into the space. This movement, which mimics wound healing is disrupted if the cells express dominant negative versions of either Rac, Cdc42 or Rho GTPases. |
| Cilia and flagella | Cilia and flagella are highly motile structures containing microtubules and dynein (- end directed motor) as their key constituents. Cilia and flagella can be distinguished by their beating patterns but are nearly identical in structure. Flagella tend to be longer than cilia. Both cilia and flagella can propel cells as they cycle rapidly beating up to 100 t/s. Co-ordinated beating of many cilia can move large cells. Alternatively, if the cells are immobilised, like epithelial cells lining an animal respiratory tract, co-ordinated beating of cilia propels liquid and particles over their apical surfaces. |
| Cilia and flagella construction | Cilia and flagella are constructed from a complex array of microtubules whose organisation is different along their length. The basal body sits in the cytoplasm of the cell and tethers and nucleates the axoneme through a region called the transitional zone. Whereas the top of the basal body has nine pairs of triplets the axoneme has nine pairs of doublets and two central MTs which are not linked. This arrangement is crucial for bending of the axoneme. Over 200 other proteins make up cilia and flagella and most of their functions are still not known. |
| Axonemal Dyneins | Axonemal Dyneins come in multiple forms that contain either one, two or three non-identical heavy chains. Each heavy chain has a globular motor domain, a "stalk" that binds to the microtubule and an extended tail (or "stem") that attaches to a neighbouring microtubule of the same axoneme. Each Dynein molecule thus forms a cross-bridge between two adjacent microtubules of the axoneme. |
| During the "power stroke" | The motor domain undergoes a conformational change that causes the microtubule-binding stalk to pivot relative to the cargo-binding tail with the result that one microtubule slides relative to the other. This sliding produces the bending movement needed for cilia to beat and propel the cell or other particles. Groups of dynein molecules responsible for movement in opposite directions are activated and inactivated in a coordinated fashion so that the cilia or flagella can bend and flex co-ordinately |
| Nexin and sliding force | The doublets are cross-linked by Nexin and make contacts to the central pair of singlet MTs through radial
spokes. These cross-links transmit a sliding force between the tubules. If an isolated axoneme is mildly proteolyzed (which removes Nexin) and ATP added, the tubules slide on each other, rather than bending. Remember dynein walks from + to - along a microtubule |
| Defining feature of intermediate filament (IF) | 310-355 residue coiled-coil domain in each IF molecule which is flanked by N- and C-terminal blobs. The base unit of the IF is a dimer where the 2 N-termini and C-termini are in close proximity and the coiled-coil region is wrapped around the other. These dimers associate laterally with each other in an anti-parallel fashion and make end-on-end contacts with other dimers where the N- and C-termini of adjacent dimers interact. This results in the formation of proto-filaments. Tetramers of proto-filaments are twisted around
each other to form the mature IF. Note that formation of the IF is due to the intrinsic properties of the IF
proteins and do not require any nucleators or adaptors. |
| Type III Intermediate filaments | In smooth muscle, Desmin binds links dense bodies together to provide a resistive force to stretching. In skeletal muscle Desminalso prevents overstretch of the sarcomere in skeletal muscle and works in conjunction with two other intermediate filament components, Synemin and Skelemin to maintain sarcomere organisation and integrity. Thick filament (Myosin II motor bundle) or thin filament (F-actin) that actually do the work of muscle contraction. |
| Type IV Intermediate filaments : Neurofilament | Neurofilaments consist of obligate heterodimers consisting of NF-L (light), NF-M (medium) and NF-H (heavy) subunits. These are required for structural support in axons and glial cells and are frequently bound to, and transported by, microtubules. Experiments in transgenic mice reveal that neurofilaments determine the correct diameter of axons, which determines the rate by which nerve impulses are propagated down axons. |
| Type V Intermediate filaments : The Nuclear Lamins | The nuclear lamina is located on the inside of the nuclear envelope and provides structural support for it. Nuclear lamins (A, B, C) form dimers in isolation. These then associate to form a meshwork. A and C lamin are splice variants transcribed from the same gene, differing at the C-terminus. The C-terminus of B lamin is covalently attached to the membrane via polyisoprenyloid lipids. |
