5. Myosin Movement on Actin Filaments PDF

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5. Myosin Movement on Actin Filaments Actin filaments, usually in conjunction with Myosin, are responsible for many types of movements in a cell: ● Myosin is the paradigm for molecular motor proteins ○ converts chemical energy (ATP) to mechanical energy, generating force and movement ● Cell division, cell crawling, some vesicle movement, and muscle contraction

CONTRACTILE ASSEMBLY OF ACTIN AND MYOSIN Notes Actin filaments can be used to promote movement with in various cells, but the movement requires a motor protein, the classic example is myosin ● Motor proteins convert chemical energy into mechanical energy using ATP an an energy source, converting to chemical energy into movement, generating a force ○ This movement is used to drive events in cell division, cell crawling, some vesicle movement, and in muscle contraction Figure 1: focussing on the top half ● one actin filament at the top and the one pair of motor proteins that are labeled myosin II What if this myosin was going to get closer to the barbed end by using ATP as an energy source? And what if, in this first example, the actin filament can’t move? ● Every motor protein will get closer to the end of their respective cytoskeletal element through one of them moving or the other. ○ In this case, if this pair of myosins is free to move and the actin filament is not, then it will look like, upon ATP hydrolysis, that myosin protein is going to walk its way, one little step at a time, like one foot in front of the other towards, in this case, the barbed end. ○ Myosin II, remember there are a lot of different kinds of myosins, they fall into classes. ■ This would be an example of a class II myosin. ■ The class II myosins move towards the barbed end of actin filaments. ■ If you were to watch this myosin interact with this actin filament and the actin filament can’t move, the myosin will get closer relative to that actin filament to the barbed end, so it will walk over to your right. Now what if the end of that myosin that isn’t attached to the actin filament was instead attached to a

vesicle of some sort, some small transport vesicle for example? ● If the situation were still the same, where the actin filament can’t do the moving, then the myosin is still going to get closer to the barbed end of that actin filament and it’s going to be carrying the vessel along with it. ● It’s going to look like it’s walking that vesicle down the actin filament towards the barbed end. Let’s say now on those tail ends of those myosins, instead of being attached to a transport vesicle, what if they are tethered to the tail ends of another pair of myosins that are facing in the opposite direction? ● Any pair of myosins exerts the same force as any other pair of myosins, so now we’re having a tug of war with ourselves and no one is going to be able to win this. Full picture Adjacent and oppositely directing myosins that are going to be getting closer to their respective barbed ends of the filaments that they have attached to. ● Myosin at bottom is attached to a different filament than the one at the top is ● Both will get closer to the barbed ends of the filament but they can’t move, there is relative movement as the actin can move ● a process known as filament sliding occurs, this is where they will pull the ends of the actin filaments in towards them, and the left and right extreme ends of this picture will move in towards each other and they will meet in the middle. Movement of myosin along actin filament: Cycle of ATP-dependent movement of myosin toward the barbed (plus) end of F-actin. 1. Beginning at any point, this is starting at the point in the cycle where myosin is moving along actin where it has grabbed firmly onto the actin filament and is not going to let go. a. This is also called the rigor confirmation, and it’s called that because this is where rigor mortis comes from. b. Rigor mortis literally means the stiffness of death, and during rigor mortis the reason why the muscles are locked in place is because the body has stopped generating ATP through oxidative phosphorylation, and you need ATP to make the myosin let go of the actin. c. First: A myosin head group latched onto an actin filament, and it isn’t letting go. Now what we would need to make this myosin let go of the actin is to bind ATP to it. A myosin head group has a nucleotide binding site, just like a G-actin monomer has a nucleotide binding site. And again, it’s going to accommodate an ATP.

2. In that myosin head group nucleotide binding site, you can either have nothing, you can have ATP, you can have ADP and phosphate, or you can have just ADP, depending on where in the cycle we are. So as we begin our trip through the cycle we have nothing in our nucleotide binding site. And that is the rigor confirmation and that head group is not going to let go of that actin filament. 3. The way to make myosin let go of the actin filament and reset, so to speak, is to bind ATP into its binding site, which you can see in the second portion of this figure. 4. When ATP occupies the nucleotide binding site of the myosin head group it causes the myosin to let go. Now the ATP in that nucleotide binding site can be hydrolyzed. That will be the next step in this cycle. As you can see working our way down the figure, ATP gets hydrolyzed into ADP and phosphate, and at the moment, both of those pieces are still in the nucleotide binding site of that myosin head group, but look at the conformational change that has been made. We have converted the chemical energy in the hydrolysis of ATP to the mechanical energy of cocking that lever arm and moving it from the position leaning to the left to, essentially, straight up and down. 5. The third portion of this figure you see the effective ATP hydrolysis. You see that now the head group of that myosin motor protein is lined up near a different monomer in that actin filament. A couple of the G-actin monomers in the filament have been highlighted in a darker red color just to show you that relative to where we began, we now are lined up a little further towards the plus end, or barbed end, of this actin filament. a. Now unlike ATP-bound myosin which has no affinity for an actin filament, ADP-bound myosin has a small affinity for actin, and it will eventually make contact with the next neighboring G-actin monomer in that filament. 6. And when it does, it’s going to grab on weakly and cause the ejection of the inorganic phosphate, as you see in the next portion of the figure. a. This forcible ejection of the phosphate group triggers what’s called the power stroke, which returns that head group back to the original confirmation that it was in before. b. But since it had previously grabbed onto a monomer in the actin filament that is closer to the plus or barbed end of the filament, you have now moved, relative to the actin filament, closer to the plus or barbed end. 7. Upon the completion of the power stroke, the ADP is ejected out, basically as the power stroke is occurring the ADP is lost and you’re right back where you started from. a. You’re in the rigor confirmation, there’s nothing occupying the nucleotide binding site, and you have moved as the green arrow indicates relative to that actin filament closer to the plus end, one step at a time. The next thing that would happen would be to bind another ATP molecule; that will cause the head group to let go. Then the ATP will be hydrolyzed, causing that lever arm to be cocked forward; then the ADP and phosphate in there makes the myosin regain some affinity for the actin so it will eventually grab on to the next G-actin molecule in line; when it makes that interaction the phosphate group is ejected; that causes the power stroke to occur; as the power stroke is finishing the ADP is lost, and once again we’re right back where we started from. Where you have a rigor confirmation, myosin grabbed tightly on to that actin filament, not going to let go until the next ATP binds there, and we have gotten one more step closer to the plus or barbed end of that microfilament.

Video Muscle myosin is a dimer with two identical motor heads that act independentlry ● Each myosin head has a catalytic core and an attached lever arm: a coiled coil rod that ties the two heads together and tethers them to thee thick filament at the top of the helical actin filament 1. Start: the myosin heads contain bound ADP and phosphate and they have a weak affinity for actin once one of the heads docks property onto an actin subunit. 2. Phosphatase is released, this releases strengthens the binding of the myosin head to actin and triggers the force generating the power stroke that moves the actin filament ADP then dissociates and ATP binds to the empty nucleotide binding site causing the myosin head to detach from the actin filament 3. On the detached head ATP is hydrolyzed which is recocks the lever arm back to pre-stroke status a. Thus like a spring the arm stores the energy released by ATP hydrolysis and the cycle can repeat after 4. The actin filament does not slide back after being released by the motor head because there are so many other myosin molecules also attached to it holding it under tension Glass slide, coverslip, myosins Myosins move towards + end or - end of actin filaments 1. Introduce myosin molecules in a liquid solution and fixed to the slide 2. Then add red flourescently labeled actin filaments a. Allow then to circulate around 3. Perhaps attaching to a head group 4. If ATP is supplied, that myosin head group will walk closer to the + end 5. The myosins don’t move to the actins do the moving, under fluorescence microscopy, we can see the actin move under imagining, looks like they’re swimming across the view

In vitro movement of actin filaments by immobilized myosin proteins: Actin and Myosin...