Role of Tubulin Post-Translational Modification in Functional Adaptation of Microtubules

University Of Georgia Research Foundation Inc, Athens GA

Investigators

Abstract

The tubulin proteins, which make up the microtubules in cells, are subjected to several types of post-translational modifications. All of the post-translational tubulin modifications are highly evolutionarly conserved, from unicellular organisms to humans. The focus of this project is to uncover the biological function of one of these modifications, the glycylation of tubulin. Tubulin glycylation is an enzymatic process that leads to addition of a number of glycine amino acid residues to specific glutamic acids of the primary peptide sequence of either alpha- or beta-tubulin. This modification has been so far found exclusively in cells which have either cilia or flagella, suggesting that it plays a role in either the formation or function of these organelles. Cilia and flagella are evolutionarly conserved motile organelles which contain eleven specialized microtubules that are essential for generating organellar movement. High speed bending motion of cilia and flagella is responsible for movement of whole cells and propagates external medium along the surface of stationary cells. Cilia or flagella are formed on the surface of many respiratory, reproductive, sensory, neural and embryonic cell types in multicellular organisms, as well as on many unicellular organisms. The ciliated protozoan, Tetrahymena thermophila, is used as a model to study the biological role of tubulin glycylation. Tetrahymena assembles about a thousand cilia on its surface. Tetrahymena tubulins are encoded by a very small number of genes, which can be manipulated by an extensive array of molecular and genetic methods. In this organism,it is now possible to either completely eliminate or mutate alpha- or beta-tubulin genes in vivo. Furthermore, even lethal or highly deleterious mutations in tubulin genes can be studied, using a unique approach of "lethal heterokaryons," which allows for maintenance of lethal mutations in the germline nucleus, and bringing them to expression in the somatic nucleus in a controlled manner. The unique features of Tetrahymena have recently allowed the Gaertig laboratory to establish that tubulin glycylation is essential for survival of this organism. They found that mutating the domain of beta-tubulin that is subjected to glycylation results in a failure of the normal organization of ciliary microtubules. Furthermore, the same mutation also affected the properties of an additional group of microtubules located near the plasma membrane, known as the cortical microtubules. Importantly, in Tetrahymena, both ciliary and cortical microtubules are subjected to glycylation. However, the same mutation of the glycylation domain of beta-tubulin affected ciliary and cortical microtubules in fundamentally different ways. While the deficiency in glycylation led to the loss of specific subtypes of ciliary microtubules, the same mutation caused an abnormal persistence of cortical MTs, which interfered with the completion of cell division. To account for the seemingly different effects of the same tubulin modification on the two different types of microtubules (ciliary and cortical), Dr. Gaertig offers three non mutually exclusive hypotheses regarding the function of tubulin glycylation and possibly other types of tubulin modifications. These are: 1), modification affects intrinsic properties of microtubules; 2), modification affects binding or activity of microtubule-associated proteins; and 3), modification affects the tubulin subunit exchange in assembled microtubules. Specific aims are designed to test predictions derived from the hypotheses. Experiments will be performed to characterize the biochemical structure of modification domains in subsets of microtubules. Several microtubule-associated proteins that localize to the cilia will be examined to determine whether their localization and activity are affected by tubulin glycylation. Conditional mutants defective in tubulin glycylation will be used to analyze the relative distribution and flux of normal and glycylation-deficient tubulin. The conditional tubulin glycylation mutants will also be used for discovery of novel proteins whose localization to cilia is dependent on tubulin glycylation. In addition to the anticipated scientific results, the project will provide a training ground for graduate and undergraduate students.