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Facilitator Questions
An in vitro motility assay allows a type of biological motion to occur outside of the cell and in a “test tube” environment. In this lecture, two in vitro motility assays are described. A purified motor is attached to an artificial bead (e.g. a 1 micron polystyrene bead) and the motor will transport the bead along a microtubule attached to a glass surface (microtubule made from purified tubulin) in the presence of ATP. In another assay, the motor is fixed onto the glass surface and the fixed motors push microtubules across the surface (like people standing in place and passing a pole from one to another). These assays are useful because you can study how these proteins work without the complications of the several thousand other proteins in the cell and one has complete control of the environment- one can change the amount of the fuel (ATP concentration), add regulatory proteins, or change the motor itself through genetic engineering.
In the in vitro gliding assay, kinesin is bound nonspecifically to the glass, facing in all directions. Why is it that the microtubules are moving continuously in one direction and are not in a tug-of-war? What end of the microtubule is “leading” (plus or minus end) as they travel across the glass? What is the gravitational attractive force produced by you and your friend at arms length and how does it compare to the force produced by one molecular motor (let us say that it is 5 pN)? How do muscle myosin and kinesin differ in their motility cycles? Explain the different functions of the “motor domain” and the “tail domain”? Which one is most highly conserved within a motor superfamily (e.g. between all kinesins or between all myosins) and why? Why is it important for the cell to organize its microtubules with uniform polarity (e.g. microtubule plus ends extending away from the centrosome? Why is muscle myosin non-processive while conventional kinesin has evolved to be processive?
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