You have purified a protein, MT1, that binds only to GTP-bound tubulin. You proc

Question

You have purified a protein, MT1, that binds only to GTP-bound tubulin. You proceed to fuse MT1 to GFP and find that the fusion of GFP to MT1 does not disrupt MT1 function in any way (good news!). You use a different dye to label the microtubules in red so that you can visualize MT1-GFP and the microtubules at the same time. (Note that the red dye does not affect the microtubule’s properties.)

A. If you were to polymerize some red-labeled microtubules in a dish, would you expect MT1-GFP to be associated with one or both ends of a microtubule, with the middle of the microtubule, or be evenly distributed throughout the microtubule? Explain. . .

B. If you were to take time-lapse images in which you visualize only MT1- GFP (and not microtubules), what would you expect to see? Describe what you think would happen to MT1-GFP over time with respect to its localization inside of this cell

Explanation / Answer

A). The MT1-GFP is expected to be associated with the either ends of the microtubules, however, the binding with alpha end is expected to be longer as compared with the beta end. MT1 protein is known to bind only the GTP-tubulin. Since GTP is present on the either ends of the polymerized tubulin GFP labelled MT1 can be seen only on the ends. The GTP stably binds to alpha-tubulin and performs structural function, whereas GTP bound to beta-tubulin hydrolyzes to GDP.

B). Occurance of MT1-GFP (but not microtublules) in time lapse experiments is an indicative of catastrophe of the polymerized microtubules, which is a result of hydrolysis of all the GTPs to GDPs and inorganic phosphates leading to the rapid depolymerization and shrinkage of the microtubules.
Over time, the microtubules would be expected to rescue i.e., tubulin would polymerize again due to phosphorylation of GDPs to GTP at the alpha and beta ends. Phosphorylation would result in addition of the MT1-GFP proteins at the either sides of the microtubules.

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