Tubulin Microheterogeneity

Further studies by Illana Gozes led to the exciting discovery that brain a-tubulin and ^-tubulin display extensive microhetero-geneity, and several nonidentical isotubulins, that differ in iso-electric points but have similar molecular weights, had been identified. The extensive microheterogeneity of tubulin appeared to be more prominent in the brain than in other tissues such as spleen or liver. Moreover, brain tubulin microheterogeneity was found to be developmentally determined, increasing in the mature brain [91].

My tubulin group, which initially consisted of Schmitt and Gozes, quickly grew to include the visiting Scientists: Annie de Baetselier (Universitaire Instelling Antwerpen), Arlette Fellous (INSERM, Bicetre, France), and Susanna Rybak (Stanford University), as well as Huub J. Dodemont (a Postdoctoral Fellow from the University of Nijmegen) and Talma Scherson (a Graduate Student).

Subsequent studies by Gozes and de Baetselier showed that some of the age-dependent variations in tubulin microhetero-geneity are controlled at the mRNA level and that there is dissimilarity in the distribution of isotubulins isolated from various regions of rat brain. These observations may reflect, to some extent, differences in tubulin microheterogeneity within the different brain-cell types [92,93]. However, multiple tubulin forms were found to be expressed by a single neuron, suggesting an additional hypothesis, namely, that the various isoforms may be differentially utilized in different subcellular structures [94].

At this stage of our work, I was fortunate to have been able to recruit Irith Ginzburg (now a Professor at the Department of Neurobiology of the Weizmann Institute) and establish with her a collaboration that lasted for ten years. It became clear that further progress in our studies required cloning of the tubulin genes and their sequence determination. What is now almost a routine exercise was in those early days (end of the 70s) a difficult and demanding task. The goal was achieved by Irith Ginzburg and her colleagues, who were able to isolate several cDNA clones bearing sequences coding for rat brain a-tubulin and ^-tubulin, as well as actin. In addition, several rat a-tubulin pseudogenes were identified and sequenced [95-97]. We were not alone in this endeavor. Simultaneous studies in many other laboratories established that a- and ^-tubulins are encoded by dispersed multigene families that are subject to differential regulation during development and differentiation. It was also observed that the large number of tubulin isotypes is due not only to expression of multiple tubulin genes, but also to various posttranslational modifications. However, the possible functions of the tubulin isotypes remains elusive (reviewed by Refs. 98,99).

We used the tubulin and actin cDNA clones to study the control of mRNA expression in a number of cell systems. Some insight into the regulation of tubulin genes was obtained from our studies with nonneuronal cells. Thus, Arlette Fellous and Irith Ginzburg showed that treatment of human lymphoblastoid cells with either a- or ^-interferon (IFN) induced a marked increase in the amounts of tubulin mRNA sequences. The specific induction of tubulin mRNA in IFN-treated cells was abolished when these cells were pretreated with colchicine, suggesting two independent regulatory sites for tubulin mRNA expression. The expression of a- and ^-tubulin did not seem to be coordinately controlled, since the treatment with IFN-6 induced a more pronounced increase in the level of a-tubulin than of ^-tubulin mRNA. It is interesting to note that the cytoplasmic actin mRNA levels were not affected following IFN treatment of these cells, indicating that the two cytoskeletal elements, tubulin and actin, are differentially regulated [85,100]. In another study, Irith Ginzburg, Susanna Rybak, and Yosef Kimhi used cDNA probes to reveal a biphasic regulation by dibutyryl cyclic AMP of tubulin and actin levels in neuro-blastoma cells. The expression of actin mRNA during the differentiation of these cells differed from that of tubulin mRNA, suggesting again that the mRNAs of these two cytoske-letal proteins are independently regulated [101]. Irith also pioneered in the use of tubulin antisense oligodeoxyribonucleo-tides to prevent neurite outgrowth in nerve growth factor induced PC12 cells. The results showed that at least two tubulin isoforms are involved in neurite outgrowth [102].

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