Analysis of properties of cilia using Tetrahymena thermophila.

Cilia and eukaryotic flagella are important structures required for the motility of cells, the movement of medium across the surfaces of cells, and the connections between the receptor and synthetic portions of sensory cells. The axoneme forms the cytoskeleton of the cilium comprising several hundreds of proteins that assemble into the 9 + 2 arrangement of outer doublet and central pair microtubules, the inner and outer rows of dynein arms, and many other structures. Tetrahymena thermophila is an excellent model organism for the study of cilia and ciliogenesis. The cell is covered by about 1,000 cilia which are essential for survival. Additionally, the Tetrahymena genome is available and targeted genetic manipulations are straightforward. In this chapter, we describe five protocols that examine properties of cilia: (a) measuring mRNA levels to see the effect of deciliation on gene expression; (b) swimming velocity and linearity; (c) ciliary length and density; (d) phagocytosis that occurs through the ciliated oral apparatus; and (e) depolarization-induced ciliary reversal.

Identification and characterization of dynein genes in Tetrahymena.

We describe the protocol through which we identify and characterize dynein subunit genes in the ciliated protozoan Tetrahymena thermophila. The gene(s) of interest is found by searching the Tetrahymena genome, and it is characterized in silico including the prediction of the open reading frame and identification of likely introns. The gene is then characterized experimentally, including the confirmation of the exon-intron organization of the gene and the measurement of the expression of the gene in nondeciliated and reciliating cells. In order to understand the function of the gene product, the gene is modified-for example, deleted, overexpressed, or epitope-tagged-using the straightforward gene replacement strategies available with Tetrahymena. The effect(s) of the dynein gene modification is evaluated by examining transformants for ciliary traits including cell motility, ciliogenesis, cell division, and the engulfment of particles through the oral apparatus. The multistepped protocol enables undergraduate students to engage in short- and long-term experiments. In our laboratory during the last 6 years, more than two dozen undergraduate students have used these methods to investigate dynein subunit genes.