Question

1. You want to understand the role of dynein in microtubule transport in a newly discovered species of parasitic fungus. To do this, you chemically induce the fusion of a dynein adaptor protein to cargo vesicles that are marked with GFP (leading to the rapid recruitment and activation of the Dynein motor complex). You notice that after several minutes these GFP- vesicles accumulate on the periphery of the cell and that by time-resolved microscopy, most vesicles seem to be moving away from the nucleus. What can you infer about the microtubule polarity of these cells?

2. You have discovered and purified a new type of microtubule-associated motor protein. You are unsure about which type of motor protein it is, however. To test this, you perform a microtubule-pelleting assay in the presence of molar excess AMP-PNPP. Upon centrifugal pelleting of your protein and microtubules, you find that this protein has pelleted with the microtubules. Is this protein likely to be a new type of dynein molecule, or kinesin?

Answer 1

Microtubule is a polymer of globular tubulin subunits. The polymers are arranged in a cylindrical manner, measuring 24nm in diameter. Also, it is a polar structure. Its polarity arises from the head-to-tail arrangement. The microtubule-organising-center helps in determining the organization of the microtubule-associated structures and organelles.
The organization of microtubule, together with its polarity, provides the navigational information needed to direct cargo to its destination. The minus (-) ends are found at the MTOC and the plus (+) ends at the periphery.
In the given scenario, the GFP-vesicles are near the periphery, and are thus moving towards the + ends of the microtubule.

The microtubule-pelleting assay allows for the identification of proteins that are bound to the microtubules. When spun at high speed, microtubules will pellet and thus, any protein bound to microtubules will also pellet with it. Kinesiin remains in continuous contact with microtubule due to catalysis of ATP. Thus, in the presence if AMP-PNP (a non-hydrolyzable ATP analogue mimicking the ATP bound state), the kinesin remains bound to microtubule.
In the given scenario, in the presence of excess of molar excess AMP-PNP and upon spinning, the pellet contains the protein and the microtubule.
Thus, the protein is a new kinesin.