Functional differences of beta-tubulin isotypes: A study of microtubule assembly and antimitotic drug resistance

Mammalian cells express 7 β-tubulin isotypes in a tissue specific manner. This has long fueled the speculation that different isotypes carry out different functions. To provide direct evidence for their functional significance, class III, IVa, and VI β-tubulin cDNAs were cloned into a tetracycline regulated expression vector and stably transfected Chinese hamster ovary cell lines expressing different levels of ectopic β-tubulin were compared for effects on microtubule organization, microtubule assembly and sensitivity to antimitotic drugs. It was found that all three isotypes coassembled with endogenous β-tubulin. βVI expression caused distinct microtubule rearrangements including microtubule dissociation from the centrosome and accumulation at the cell periphery; whereas expression of βIII and βVIa caused no observable changes in the interphase microtubule network. Overexpression of all 3 isotypes caused spindle malformation and mitotic defects. Both βIII and βIVa disrupted microtubule assembly in proportion to their abundance and thereby conferred supersensitivity to microtubule depolymerizing drugs. In contrast, βVI stabilized microtubules at low stoichiometry and thus conferred resistance to many microtubule destabilizing drugs but not vinblastine. The 3 isotypes caused differing responses to microtubule stabilizing drugs. Expression of βIII conferred paclitaxel resistance while βVI did not. Low expression of βIVa caused supersensitivity to paclitaxel, whereas higher expression resulted in the loss of supersensitivity. The results suggest that βIVa may possess an enhanced ability to bind paclitaxel that increases sensitivity to the drug and acts substoichiometrically. At high levels of βVIa expression, however, microtubule disruptive effects counteract the assembly promoting pressure exerted by increased paclitaxel binding, and drug supersensitivity is lost. From this study, I concluded that β-tubulin isotypes behave differently from each other in terms of microtubule organization, microtubule assembly and dynamics, and antimitotic drug sensitivity. The isotype composition of cell can impart subtle to dramatic effects on the properties of microtubules leading to potential functional consequences and opening the opportunity to exploit differences in microtubule isotype composition for therapeutic gain. ^