Direct Comparison of Glu and Tyr Tubulin Distribution in Mitotic Spindles

The C-terminus of a-tubulin undergoes a reversible posttranslational tyrosination/detyrosination. The distributions of the tyrosinated (Tyr) and nontyrosinated (Glu) species during mitosis of cultured cells have been investigated by immunofluorescence using antibodies directed against the C-terminus of either Tyr or Glu tubulin. The distribution of Tyr tubulin differed from that of Glu tubulin at each stage of mitosis; in general, the distribution of Tyr tubulin was similar to that of total tubulin, whereas Glu tubulin had a more restricted distribution. The Glu species was found in half-spindle fibers but was not detected in astral fibers at any stage and was seen in the interzone only during telophase. These results were confirmed by a direct comparison of the distributions of Tyr and Glu tubulin in cells double-labeled with the two antibodies. Evidence for the occurrence of Tyr and Glu tubulin in each class of half-spindle fibers (kinetochore and polar) was obtained from the staining patterns of the two antibodies in cold-treated cells. Immunoblots of extracts prepared from synchronous mitotic cells showed that Glu tubulin was a minor species of the total tubulin in the spindle; no changes in the amount of either Tyr or Glu tubulin were detected at any stage of mitosis. These results show that Tyr tubulin is the major species in the mitotic spindle and is found in all classes of spindle fibers, whereas Glu tubulin is present in small amounts and shows a more restricted distribution. The presence of two biochemically distinct forms of alpha-tubulin in the spindle may be important for spindle function. T HE mechanism of chromosome alignment and subsequent separation during cell division has long fascinated cell biologists. In eukaryotes, a complex structure, the mitotic spindle, is elaborated to effect this chromosome segregation. Studies of the ultrastructure of the mitotic spindle, the dynamics of the spindle during mitosis in living cells, and the effects of various drugs or physical agents on spindle organization have characterized the changes in organization that the microtubules, the constituent elements of the spindle, undergo during mitosis (these topics are subjects of recent reviews: see references 17, 25, 38, 42, and 46). These studies have also clearly established a role for microtubules in chromosome segregation, and coupled with knowledge of the in vitro properties of tubulin (reviewed in references 39 and 47), have led to several models for mitosis (4, 24, 34, 35). Nonetheless, all of these models avoid one of the critical questions raised by studies of the rearrangement of microtubules during mitosis; namely, what is the molecular nature of the control that permits some microtubules to shorten (e.g., kinetochore microtubules during anaphase), while at the same time and virtually in the same place, other microtubules are growing (e.g., interzonal microtubules during anaphase). Explanations based on local changes in the intracellular milieu, such that microtubule polymerization or depolymerization is favored, implicate specialized zones around each spindle microtubule; such mechanisms are hard to envision, much less test. One possibility that might explain the differences in behavior of individual spindle microtubules is that their component proteins are different. This is essentially the "multi-tubulin hypothesis" (18) applied to the mitotic spindle, although currently there is no evidence for different tubulins within the spindle. Nonetheless, considerable evidence exists for extensive tubulin heterogeneity in the brain (16, 19, 20) and even within a single neuron (21). Similarly, differences at the molecular level have been described between the a-tubulin incorporated into sea urchin flagella and cilia and that present in the cytoplasm (53). This later study provided an even more striking result: the Aand B-subfibers of the axoneme contained different species of tubulin. Thus, there is evidence from some systems that tubulin heterogeneity exists not only within a single cell but within a single organelle. One possible source for this tubulin heterogeneity is that there are different genes that specify different tubulin proteins (isoforms). Studies of the genetic organization of tubulin have shown that there are at least four different isoforms of both aand ~-tubulin in both vertebrates and invertebrates (11, 44). Tubulin genes have been shown to be expressed in a developmental and tissue-specific manner, although in general more than one isoform is expressed in each tissue. Mutations in the testis-specific ¢/-tubulin of Drosophila lead to abnormal meiosis and impared axonemal formation, which suggests that a single isoform can perform multiple microtubule functions (26, 27). For those cases in which multiple © The Rockefeller University Press, 0021-9525/86/03/1118/09 $1.00 1 118 The Journal of Cell Biology, Volume 102, March 1986 1118-1126 isoforms are expressed, it is not known whether the different isoforms have distinct localizations or functions. Although genomic complexity can provide for some heterogeneity, it clearly cannot account for all of it. In the brain, as many as 18 isoelectric variants of tubulin have been detected (16). Posttranslational modifications are likely to be the source of this additional heterogeneity. Several posttranslational modifications of tubulin have been described, which include phosphorylation of the B-subunit (15), and acetylation (33) and tyrosination (5, 6) of the a-subunit. Of these three, tyrosination is unique to tubulin. It is a reversible reaction and results in the addition (or removal) of a tyrosine residue from the C-terminus of a-tubulin (2). The reaction occurs in both vertebrates (43) and invertebrates (28) and is thus a good candidate for a modification involved in a ubiquitous microtubule function, such as mitosis. An aid to our understanding of the function of tubulin posttranslational modifications would be a determination of the intracellular location of the modified and unmodified species. In an earlier report, we described the preparation of peptide antibodies specific for the C-termini of tyrosinated (Tyr) l and nontyrosinated (Glu) a-tubulin and found that Tyr and Glu tubulin are largely segregated into different interphase microtubules (22). In this paper, we describe the distribution of these species of a-tubulin during mitosis. Materials and Methods