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Abstract Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to textbook models of tubulin self-assembly, we have recently demonstrated that microtubules elongate by addition of bent guanosine triphosphate tubulin to the tips of curving protofilaments. Here we explore this mechanism of microtubule growth using Brownian dynamics modeling and electron cryotomography. The previously described flaring shapes of growing microtubule tips are remarkably consistent under various assembly conditions, including different tubulin concentrations, the presence or absence of a polymerization catalyst or tubulin-binding drugs. Simulations indicate that development of substantial forces during microtubule growth and shortening requires a high activation energy barrier in lateral tubulin-tubulin interactions. Modeling offers a mechanism to explain kinetochore coupling to growing microtubule tips under assisting force, and it predicts a load-dependent acceleration of microtubule assembly, providing a role for the flared morphology of growing microtubule ends. 

Glioblastoma ranks among the most lethal of primary brain malignancies, with glioblastoma stem cells (GSCs) at the apex of tumor cellular hierarchies. Here, to discover novel therapeutic GSC targets, we interrogated gene expression profiles from GSCs, differentiated glioblastoma cells (DGCs), and neural stem cells (NSCs), revealing EYA2 as preferentially expressed by GSCs. Targeting EYA2 impaired GSC maintenance and induced cell cycle arrest, apoptosis, and loss of self-renewal. EYA2 displayed novel localization to centrosomes in GSCs, and EYA2 tyrosine (Tyr) phosphatase activity was essential for proper mitotic spindle assembly and survival of GSCs. Inhibition of the EYA2 Tyr phosphatase activity, via genetic or pharmacological means, mimicked EYA2 loss in GSCs in vitro and extended the survival of tumor-bearing mice. Supporting the clinical relevance of these findings, EYA2 portends poor patient prognosis in glioblastoma. Collectively, our data indicate that EYA2 phosphatase function plays selective critical roles in the growth and survival of GSCs, potentially offering a high therapeutic index for EYA2 inhibitors. 

How temperature specifically affects microtubule dynamics and how these lead to changes in microtubule networks in cells have not been established. We investigated these questions in budding yeast, an organism found in diverse environments and therefore predicted to exhibit dynamic microtubules across a broad temperature range. We measured the dynamics of GFP-labeled microtubules in living cells and found that lowering temperature from 37°C to 10°C decreased the rates of both polymerization and depolymerization, decreased the amount of polymer assembled before catastrophes, and decreased the frequency of microtubule emergence from nucleation sites. Lowering to 4°C caused rapid loss of almost all microtubule polymer. We provide evidence that these effects on microtubule dynamics may be explained in part by changes in the cofactor-dependent conformational dynamics of tubulin proteins. Ablation of tubulin-binding cofactors (TBCs) further sensitizes cells and their microtubules to low temperatures, and we highlight a specific role for TBCB/Alf1 in microtubule maintenance at low temperatures. Finally, we show that inhibiting the maturation cycle of tubulin by using a point mutant in β-tubulin confers hyperstable microtubules at low temperatures and rescues the requirement for TBCB/Alf1 in maintaining microtubule polymer at low temperatures. Together, these results reveal an unappreciated step in the tubulin cycle.