Generation of antisera that discriminate among mammalian α-tubulins: Introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function

Abstract

To assay the functional significance of the multiple but closely related α-tubulin polypeptides that are expressed in mammalian cells, we generated three specific immune sera, each of which uniquely recognizes a distinct α-tubulin isotype. All three isotypes are expressed in a tissue-restricted manner: one (Mα3/7) only in mature testis, one (Mα4) mainly in muscle and brain, and the third (Mα6) in several tissues at a very low level. A fourth specific antiserum was also generated that distinguishes between the tyrosinated and nontyrosinated form of a single α-tubulin isotype. Because individual tubulin isotypes cannot be purified biochemically, these sera were raised using cloned fusion proteins purified from host Escherichia coli cells. To suppress the immune response to shared epitopes, animals were first rendered tolerant to fusion proteins encoding all but one of the known mammalian α-tubulin isotypes. Subsequent challenge with the remaining fusion protein then resulted in the elicitation of an immune response to unique epitopes. Three criteria were used to establish the specificity of the resulting sera: (a) their ability to discriminate among cloned fusion proteins representing all the known mammalian α-tubulin isotypes; (b) their ability to uniquely detect α-tubulin in whole extracts of tissues; and (c) their capacity to stain microtubules in fixed preparations of cells transfected with sequences encoding the corresponding isotype. The transfection experiments served to demonstrate (a) the coassembly of Mα3/7, Mα4, and Mα6 into both interphase and spindle microtubules in HeLa cells and NIH 3T3 cells, and (b) that the Mα4 isotype, which is unique among mammalian α-tubulins in that it lacks an encoded carboxy-terminal tyrosine residue, behaves like other α-tubulin isotypes with respect to the cycle of tyrosination/detyrosination that occurs in most cultured cells.