Microtubule Stability: A Comparison Between ATP Depleted Cells and Taxol Treated Cells

Microtubules (MTs), composed of α/β tubulin dimers, play a crucial role in cell division, cell polarity, and intracellular transport. In cells, they undergo “dynamic instability”, a process whereby microtubules randomly switch between polymerization, depolymerization and stasis, in which case, these MTs are called “stable.” It’s believed that both microtubule-binding proteins and changes in tubulin post-translational modifications (PTMs) can alter the percent of stable microtubules in cells. MTs in cells treated with the anti-cancer drug, taxol, become ‘hyper-stable,’ less dynamic, and resist depolymerization by pharmacological agents such as nocodazole. Interestingly, when cells are depleted of ATP, they also accumulate hyper-stable MTs that are resistant to depolymerization. The mechanism of action of taxol has been heavily researched but is still not completely understood. In contrast, little is known about how ATP levels influence MT stability. The goal of this study was to compare the MT distribution in cells depleted of ATP and cells treated with taxol in the presence of depolymerizing drugs, and in particular, to compare the recovery of MT dynamic instability upon drug removal. PtK2 rat kangaroo kidney epithelial cells were treated with either taxol or ATP-depleting drugs (sodium azide plus 2-deoyxglucose), and then challenged with nocodazole, a potent MT-depolymerizing drug. As expected from prior research, taxol and/or ATP depletion results in many ‘hyper-stable’ MTs that are resistant to depolymerization. Upon washing out the taxol or ATP-depleting drugs, the microtubules regained their sensitivity to nocodazole and began to depolymerize. We next did a careful time course to determine whether the stabilization of MTs is reversible upon drug removal and if so, to determine when the cells’ MTs regained sensitivity to nocodazole.  We found that the effects of both drugs were reversible. MTs regained their sensitivity to nocodazole within 30 minutes after taxol wash-out, and within 10 minutes after removal of ATP-depleting drugs. This was determined by examining the MTs using immunofluorescence microscopy. The fact that the refractory period (the point at which MTs regain sensitivity to nocodazole) is different suggests that the mechanisms by which MTs are stabilized differs. Additional replicate experiments will be done and quantified to see if the difference in refractory periods are statically significant.