Dye-feeding assay of Tetrahymena, for studying the effects of cigarette smoke and E-cigarette liquid components on ciliary activity

Adapted from: Phagocytosis in Tetrahymena as an Experimental System to Study the Toxic Effects of Cigarette Smoke. Kenyon College, Biol 09; Smith, J.J., Wiley, E., Cassidy-Hanley, D.M. 2012. Tetrahymena in the Classroom. Methods in Cell Biology; Bozzone, D.M. and Martin, D.A. 2000. An Experimental System to Study Phagocytosis. www.ableweb.org.

Overview

Experiment Proposal assignment.

With input from your TA, you will design two experiments that test the effect of cigarette smoke and/or E-cigarette liquid components on the activity of Tetrahymena cilia. You will perform these experiments during the Project weeks, and then each member will prepare a Project Report based on the results.

Background

When hungry Tetrahymena encounter a food source such as bacteria, they use their cilia to sweep it toward the cytostome so that it can be ingested by phagocytosis. This process can be visualized in the laboratory by feeding stained yeast cells, India ink particles (Keenan, 1984; Bozzone, 1998), or the pigmented algae Chlorella to Tetrahymena.

We will be using India ink as Tetrahymena “food.” The vacuoles that form are full of black ink particles and therefore are easy to identify. The movement of the vacuoles inside the cell as new ones form can also be observed. If you are sufficiently patient, sometimes the vacuoles can be observed “voiding” their contents from the cytoproct into the extracellular space. In addition to phagocytosis, ciliary motion and swimming behaviors are easier to observe when Tetrahymena are suspended in a dilute ink suspension.

Overview of  Projects

As you have (hopefully) seen, Tetrahymena readily phagocytose ink particles. The number of vacuoles present is related to the amount of time the organisms are allowed to feed, provided the time interval is less than 30 minutes. After 30 minutes, cells begin expelling the residual contents of food vacuoles via the cytoproct. Phagocytosis can be used as a way of indirectly assaying the effects of various substances on ciliary activity, since this is required for entry of particles into the oral groove. If a substance added to Tetrahymena cells inhibits their ciliary motion, fewer food vacuoles will form over time since less ink will be ingested by the cells.

As we noted earlier, cilia are found on the surfaces of most cells in mammals. Motile cilia are found only on specialized cells, including those that line the respiratory tract. These cilia have a rhythmic beating pattern that allows them to clear the airways of mucus and debris. Non-motile (primary) cilia are found on the surface of almost all mammalian cells and function as signal receptors for relaying information. For example, primary cilia in the kidney bend in response to urine flow, which is transduced as a signal that allows flow to be adjusted via feedback loops.

Effects of extracts from cigarette smoke and E-cigarette liquid on food vacuole formation

Smoking cigarettes has a well-known negative effect on the health of cilia in the respiratory tract. Exposure to toxic chemicals and tar resin in smoke damages the cilia, inhibiting their ability to clear mucus and debris and prevent it from being trapped inside the lungs. Over time, the effects of reduced mucus clearance lead to “smoker’s cough,” and may progress to emphysema.

E-cigarettes are a relatively new invention that were designed to circumvent some of the negative health effects of smoking. The devices use a metal element to heat “e-liquid” containing nicotine, propylene glycol, glycerin, and flavorings. When the e-liquid is heated, an aerosol (vapor) is produced that can be inhaled, similar to smoke from a cigarette. Although E-cigarettes are commonly portrayed as being harmless, or a safer alternative to smoking, few studies have been done to test the effects of inhaling this vapor into the lungs. The vapor might contain toxins and metal contaminants produced during the heating of the liquid. Additionally, the flavoring compounds added to popular e-liquid brands have not been tested for ciliary toxicity.

In your  Project, you  will design and carry out an experiment to test the effects of cigarette smoke extracts and/or E-cigarette liquid on the ciliary activity of Tetrahymena. You will have the materials and experimental conditions listed below at your disposal. First, develop a hypothesis that you want to test, and then design an experiment to test this hypothesis. Discuss your ideas with your group, and then complete the first Experiment Proposal together.