Lecture 21 Actin Motors & Regulators PDF

In this lecture, we will investigate myosins, their mechanochemical cycle, how they move on actin filaments – looking at nonmuscle myosins, and muscle myosin. Myosin contains an actin binding motor domain that binds ATP and hydrolyses it to ADP – moving through a mechanochemical cycle of binding an...

In this lecture, we will investigate myosins, their mechanochemical cycle, how they move on actin filaments – looking at nonmuscle myosins, and muscle myosin. Myosin contains an actin binding motor domain that binds ATP and hydrolyses it to ADP – moving through a mechanochemical cycle of binding and releasing from the F-actin filament, and coordinating stepping of two motor heads. Shown above is the domain architecture of a full length myosin chain – and a structure of the motor and neck region below. The ATP hydrolysis cycle causes movement in the converter region that is relayed to the lever arm and neck, creating a power stroke that moves the dimerization domain (tail region) relative to the motor head. This moves the rear head to the front position along F-actin. In the neck region are multiple IQ motifs. Calmodulin (CaM), also called myosin light chain – binds and stabilizes this region, making it rigid. The CaM light chains and the length of the neck are one component that determines the step size. Effectively, this is how long the motor’s legs are before it goes into the dimerization coiled coil domain. Compare and contrast this architecture with kinesin. Detailed here is the mechanochemical cycle of myosin. Things to note: look at the nucleotide state of the motor domain, look at whether the motor head is bound to the filament or not, and look at what the position of the green lever arm is. The lever arm position relates to the power stroke or recovery stroke. Note that myosins can either be in filaments (myosin II – both muscle and non- muscle myosin II), as well as dimeric forms for the unconventional myosins. Shown here is just the mechanochemical cycle for one motor head – so consider what would be happening if there is a second head for a dimeric motor – the other head would be swung from the rear position to the front position. The mechanochemical cycle of the two heads has to be coordinated for there to be processivity (continuous hand- over-hand walking along a F-actin filament). Non-muscle myosin II can be activated by phosphorylation, leading to formation of a bipolar filament. The filament can engage F-actin filaments, leading to contractile forces. Many of the functions of non- muscle myosin II are shown here. The bipolar motor filament can engage F-actin structures leading to contractile forces. https://link.springer.com/article/1 0.1007/s00018-012-1002- 9/figures/4 There are many non-muscle myosins. Aside from myosin II, many form dimers of various structures that can walk differently along actin filaments. One motor in particular, is unique: Myo VI, as it can walk to the F-actin minus end (the pointed end) – the reverse direction of all the other motors (all others walk to the plus end or barbed end of the actin filament). Different motors are expressed for different functions, cell types, cellular localization, and cargo binding/transport activities. Example: Myo X moves cargo to the tips of filopodia. Example: In the stereocilia that we use in our inner ears to detect sound, Myo III and XV transport cargo the to stereocilia tip, while Myo VI transports cargo back out. Flux in the actin filaments (treadmilling, also serves to move material out of the stereocilia). Kymography can be used to examine motor movement along an actin filament, and determine polarity of movement. An in vitro study: Single motor dimers, labeled with a green fluorophore are shown processing along an actin filament. Above: Myosin 5 (V) moves in one direction (towards the filament plus end). Myosin 6 (VI) moves in the opposite direction, towards the filament minus end. Myosin II makes a power stroke towards the barbed (plus) end of about 5 nm. Myosin V has lots of IQ motifs, and thus a longer lever arm – its power stroke moves approximately 36 nm – also towards the plus end. In contrast, the converter region of Myosin VI operates in a different way, and undergoes a distinct conformational change: it starts with the lever arm in the forward position, and then moves it to the rear position, effectively resulting in a 11 nm power stroke towards the filament minus end. Myosins take different steps along an F-actin filament. See how the Myosin V dimer movement in the lever arm moves the rear head to the forward position. The long, stable IQ motifs bound by CaM create a rigid-like neck and stalk that connect to the dimerization domain. When a myosin dimer is labeled with a fluorophore on only one of the motor heads (not both), the step size of one motor can be monitored. See the distribution of step sizes at right – average = 73.75 nm. This is because a lever arm movement of ~36 nm results in twice the movement of one motor head. This means that the trace is only monitoring the position of one motor (or foot). Not shown is the position of the other foot which needs to walk out of phase with the other, also taking ~73 nm steps. Hmmm – consider the actin filament structure. Every 36 nm corresponds to the half turn of the actin filament. This means that if a motor takes 72 nm steps, it can stay on one side of the filament. A longer or a shorter step would require that the myosin motor rotate around the filament – which could complicate cargo transport. There is play in the step size, and as we mentioned previously, the structure of the actin filament can change depending the factors that are bound, the strain the filament is under, and nucleotide state of the F- actin. The three kinds of muscle are shown. Skeletal muscle and cardiac muscle are striated, while smooth muscle is not. While we control our skeletal muscle, our smooth muscle and cardiac muscle runs on autopilot! Skeletal muscle and cardiac muscle have a common striated architecture that will be activated in similar ways by calcium, but will use distinct upstream neuronal activation pathways. In contrast, smooth muscle, which is not striated, will use a distinct calcium-dependent activation pathway. All three muscle tissues will use bipolar myosin filaments to slide against F-actin. Muscle myosin is also a nyosin II class motor – like non-muscle myosin II, it also forms bipolar filaments. Dimers of muscle myosin II become arranged in bipolar bundles called thick filaments (based on their density in EM images, relative to actin fibers in the muscle- which are termed thin filaments). The bipolar nature of these thick filament orient clusters of the motors in two opposite directions. These motor clusters will engage with actin thin filaments, oriented in opposite directions, and exert power strokes towards the thin filament plus ends. (Myosin II motors are plus end directed motors). Tarantula myosin thick filaments https://www.nature.com/articles/natu re03920 https://www.sciencedirect.com/scienc e/article/pii/S0959440X06000443 Muscle fiber cell is indicated. Each myofibril is made of many co- linear sarcomeres. Many myofibrils function in a muscle fiber. These are multinucleated cells – they are large cells formed when myoblasts fuse together. Note the mitochondria – which are required to produce the large amounts of ATP needed for the motors. This is the general structure, now let’s zoom in, before we zoom back out. In the sarcomere, myo II bipolar filaments are anchored at the M- line. The myo II filament is referred to as the thick filament. The thin filaments are the actin filaments that are polarized and have their plus ends anchored at the Z-disc. https://www.sciencedirect.com/sci ence/article/pii/S0959440X06000 443 The region marked A, constitutes half of the A-band. The region marked I constitutes half of the I- band (see next slide for full band delineation). Myo II filaments are anchored at the M-line by myomesin I and the M protein. The Myo II filaments are also attached to the Z-disc by titin – which is flexible. https://www.researchgate.net/figure/ a-Electron-micrograph-of-a-skeletal- muscle-sarcomere-demonstrating- thick-and-thin_fig16_230733216 Multiple sarcomers are indicated, all arrayed in a lattice-like structure within the myofibril. A- band, I-band, M-line, and Z-disc are indicated. The lattice like arrangement means that contraction will occur along a common axis for all the sarcomeres, generating a large muscular contraction. Myo II movement; the bipolar heads ”walk” along the opposing F-actin filaments – moving towards the filament plus ends which are anchored at the Z-discs. Because the myosin can’t actually move, it pulls on the actin, moving the Z- discs closer to the M-line. When the myosin heads release the actin, the sarcomere reverts to the relaxed state. Myo II movement; the bipolar heads ”walk” along the opposing F-actin filaments – moving towards the filament plus ends which are anchored at the Z-discs. Because the myosin can’t actually move, it pulls on the actin, moving the Z- discs closer to the M-line. When the myosin heads release the actin, the sarcomere reverts to the relaxed state. The myosin mechanochemical cycle. Note that for the bipolar filaments, many heads will be working on F-actin, generating large amounts of force. In the diagram above, note the change in position of the myosin motor head (due to lever arm movement) relative to the actin filament, which is dependent on the nucleotide- bound state of the motor (at top: myo II is bound to the red actin at left, below it is bound to the red actin at right). The net movement is for the myosin to “walk” to the plus end. Because the myosin is anchored, this force causes the actin to move towards the M-line, leading with its minus end. Good question! That’s right, the nucleotide-free state. When we exhaust our ATP, we will enter into rigor mortis. Calcium does! Contraction in striated muscle (skeletal and cardiac): A neuron will activate the muscle cell via acetylcholine release. This results in an electrical impulse that is propagated deep into the large muscle cell by T-tubules. The impulse triggers the release of calcium from the sarcoplasmic reticulum (which is part of the ER), into the sarcomeres. Contraction in striated muscle (skeletal and cardiac): Wrapping around the F-actin filaments in the sarcomere is a protein tropomyosin (this is a structural protein, not a myosin motor), which is complexed with troponin. Tropomyosin adopts two conformations – one of which occludes (blocks) the site on F- actin that myosin motor domains bind. In the relaxed conformation, myosin II cannot engage F-actin because tropomyosin blocks it. However, when calcium release is triggered, it enters the sarcomere, binds to troponin, and causes a conformational change in the tropomyosin-troponin complex, altering its binding mode with F-actin. Thus, when troponin binds calcium, tropomyosin moves to a different position on the filament, enabling the myosin II motors to gain access to their binding sites on the F-actin filament (black dots along the F-actin structure shown above), leading to contraction. During this time, calcium pumps are working to pump calcium out, and back into the sarcoplasmic reticulum. When calcium levels fall, the system reverts to the relaxed state (tropomyosin binds and occludes the myosin II binding site on F-actin). While smooth muscle does not have striations, it still functions in a similar way: bipolar filament (thick filament) sliding against F- actin thin filaments. To enable this, F-actin is anchored in different ways throughout the cell: example, attached to the extracellular matric by integrins and focal adhesions complexes, and to dense bodies that also severe as anchor points for a mesh-like structure formed by intermediate filaments. In smooth muscle, calcium is also an activator of muscle contraction, but in different mode of action than in muscle: Myosin light chain kinase (MLCK - a kinase) is activated by calcium-bound calmodulin (CaM). MLCK then phosphorylates the myosin light chain, which in turn activates the myosin motor, causing contraction. This is then turned off (leading to relaxation) by a phosphatase.