2) In class we looked at one modern method for examining microtubule dynamics in

Question

2) In class we looked at one modern method for examining microtubule dynamics in vivo, which is to use Microtubule end binding proteins like EBI, a protein that binds to the GTP-bound tubulin filaments. a. [4pts) Based on the model of Dynamic Instability, if you watched the mitrotubules in a cell in GO over time using a red fluorescent version of tubulin, and a green fluorescent version of EBI what would you expect to see? b. (4pts) If this particular cell had a lot of microtubule treadmilling rather then just dynamic instability how would your answer above change? c. (4pts) Regardless of the dynamics of your microtubules, if you added a drug that inhibited the activity of kinesin, the plus-end directed microtubule motor protein, what types of effects might you expect to see in a secretory cell in GO?

Explanation / Answer

a. In G0 phase the cells are in queiscent stage and is also called post mitotic stage, where cells do not divide. Both red fluorescent tubulin and green fluorescent EBI would not be moving much as the rate of depolymerization/polymerization is considerably controlled during this process. There will be basal level of movement of EBI and tubulin as shown by fluorescence although it will be less as cells are in a resting phase compare to cells that are actively dividing.

b. Dynamic instability of microtubules comprises of assemby/disassembly only at one end. Thus one would expect continuous polymerization and depolymerization taking place at one end (mostly plus ends). On the other hand, treadmilling would result in polymerization/depolymerization at both ends depending upon critical concentration of certain molecules. Treadmilling would result in red and green fluorescence shown at both ends of microtubules as the process is regulated by concentration of few microtubule associated factors . A beautiful visual within the cell where the fluorescence wil keep shifting from one end to other end depending upon the origin and continuation of treadmilling.

c. If one adds a drug say nocodazole or vincristine to block polymerization, one would not see a dynamic process, rather a static localisation of tubulin and EBI would be observed. However, the fluorescence of tubulin (red color) would go down as depolymerization of microtubules will be active (thus a reduction in fluorescence movement).

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