Microtubules are ubiquitous eukaryotic structures, built of dimers of alpha- and beta tubulin. They form the framework of many essential organelles, including centrioles, cilia, and the mitotic spindle. Not much is known about how cells build and maintain complex organelles made of microtubules. In this project, Dr. Gaertig and his colleagues will explore the hypothesis that cells use a set of post-translational tubulin modifications (PTMs) to mark the surface of microtubules. The idea is that these marks represent the "microtubule code," a set of structural signals that direct binding and activity of proteins to microtubules. Dr. Gaertig has been studying the function of the so-called polymodification PTMs (tubulin glutamylation and glycylation), using the ciliated protozoan, Tetrahymena, as a model. These PTMs are evolutionarily conserved and abundant in vital organelles such as cilia and centrioles. The Tetrahymena model offers a powerful combination of genetic, biochemical, and cell biological approaches.
Based on genetic data, Dr. Gaertig has identified a known microtubule severing factor, katanin, as a potential major effector protein that can "read" the polymodification marks on microtubules. A mutation in the catalytic subunit of katanin phenocopied the mutation of the polymodification region on beta-tubulin. Both types of mutants are nearly indistinguishable from each other. They have excessively long cortical microtubules that obstruct the cleavage furrow, and they assemble only short paralyzed cilia which lack the central pair. Dr. Gaertig hypothesizes that in the cell body, polymodified cortical microtubules are preferentially severed by katanin to maintain their proper length via treadmilling, and that katanin participates in the production or assembly of ciliary tubulin precursors. This research project seeks to determine the function, mutual interactions and localizations of two catalytic and a single noncatalytic subunit of katanin in Tetrahymena. Gene knockouts and conditional alleles will be used to evaluate the role of katanin at multiple stages of ciliogenesis. Biochemical methods will be used to purify katanin complexes and to discover novel katanin-binding proteins. Purified katanin will be used to test whether its activity can be affected by the levels of glutamylation in an in vitro microtubule severing assay.
Intellectual Merit: This project will study the role of a conserved microtubule severing factor, katanin, in the formation and maintenance of important eukaryotic organelles, cilia. The results should shed new light on whether and how tubulin glutamylation regulates katanin.
Broader Impact: This project will provide a training platform for 3 graduate Ph.D. students and a group of undergraduate students working on projects in Dr. Gaertig's laboratory, including some who are members of groups that are under-represented in science.
Microtubules are protein fibers that provide structural support, produce forces and establish transport routes for numerous components of cells. Microtubules are part of an internal skeleton of cells (cytoskeleton) and are critical for shaping cells and generating their motility. Microtubules are also the main component of thin projections of cells called cilia. Cilia are widely present in diverse organisms, including mammals, where they are present on most cell types. Cilia have important sensory and motility functions. Rapid beating of cilia generates motility of cells and propagates fluids along the surface of stationary cells. Inside the human body, cilia are essential for embryonic development and mutations in ciliary proteins cause a large number of diseases called ciliopathies. Cilia are composed of tubulin proteins, the main building block of microtubules, and a few hundred of other mostly highly conserved proteins. Tubulin and associated proteins form the axoneme, a stiff core structure of cilia that has 11 microtubules arranged in a highly reproducible circular pattern known as 9+2 (9 microtubules at the periphery and 2 in the middle). How tubulin and other proteins are assembled into the 9+2 axoneme is not well understood. Using a ciliated genetic model organism, a free living ciliate Tetrahymena thermophila, we found that the process of assembly of cilia requires the activity of katanin, a conserved protein complex known to sever microtubules. Surprisingly, without katanin, ciliary axonemes are too short and lack a subset of microtubules. In the absence of katanin, ciliary and non-ciliary microtubules accumulate biochemical marks formed by modified amino acids within the protein sequence (lysine acetylation, glycylation and glutamylation). We also investigated the ciliary function of kinesin-13, a protein that shortens microtubule ends. We found that kinesin-13 is required for regulation of the length and motility of cilia. Without kinesin-13, cilia assemble correctly but remain short and beat slowly. Furthermore, we found that as is the case of katanin-deficient cells, cells lacking kinesin-13, have excessive levels of biochemical marks on ciliary and non-ciliary microtubules. All these observations indicate that microtubule remodeling proteins (both shortening and severing) play a key role in the assembly of cilia, possibly by limiting the density of marks on microtubules. We also investigated the mechanisms that deposit marks on microtubules. We contributed to the discovery of a number of enzymes that are responsible for generation of biochemical marks on microtubules including enzymes that attach amino acids to tubulin: E-ligases (glutamic acid ligases) and G-ligases (glycine ligases). We also discovered the long sought enzyme that acetylates tubulin. We showed that some of the tubulin-marking enzymes regulate ciliary motility and affect cilia length. In one case, we showed that a particular tubulin modifying enzyme, TTLL6, regulates the activity of a subset of motor proteins (inner dynein arms), that generate the beating of cilia. In addition to the scientific outcomes, this project provided professional training activities for undergraduate and graduate students as well as a high school teacher.
