An Improved Quantitative Assay for Chemokinesis in Tetrahymena

Abstract

This paper presents a quantitative and sensitive assay for the measurement of chemosensory behavior in Tetrahymena. The two-phase assay is easy to perform in large quantities, so a variety of compounds can be screened under comparable conditions. A suspension of 2 x 10(5) cells ml-1 (the upper phase) is starved for 20-40 h and then gently placed on top of a 5% solution of Metrizamide (the lower phase) in a disposable microcuvette. The optical density of the lower phase is monitored at 600 nm with an automated spectrophotometer at selected time points. Optimum sensitivity of the assay is achieved when the cells slowly but continuously enter the lower phase, so that about 5% of them will be in the lower phase within 30 min. Optimal chemosensory responses occurred in Tetrahymena thermophila at about 25 degrees C. The response was delayed at 15 degrees C and markedly reduced at 35 degrees C. The data suggest three bases for quantifying the response in the assay: (1) initial slope of the absorbance versus time; (2) final maximal absorbance within the time period of measurement; and (3) signal-to-noise ratio (S/N) at a fixed time. We have quantified--in terms of S/N--the chemosensory responses in Tetrahymena for the following compounds: beta-endorphin, fibroblast growth factor, insulin, and platelet-derived growth factor (PDGF); these substances were active in nanomolar concentrations, and the maximal S/N was between 3 and 5.1. Acetylcholine was active only in millimolar concentrations; maximal S/N was 4.1 at 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemoattraction to lysophosphatidic acid does not require a change in membrane potential inTetrahymena thermophila

“…Two different assays were used to examine the chemoattraction phenotypes. The first was the two‐phase assay performed essentially as described by Koppelhus et al (1994). To set up the assay, the test compound was dissolved in the 10 mM Tris buffer with 3% nycodenz (w/v) added at the bottom of a cuvette and zeroed in the spectrophotometer.…”

“…It is interesting that many of these putative GPCRs have considerable homologies to those proposed in plants (Lampert et al, 2009), suggesting that some of the ciliate GPCRs may be plant‐like in their origins (Archibald, 2008). There are many examples of chemosensory responses in Tetrahymena (Csaba, 1985; Hellung‐Larsen, 1986; Koppelhus et al, 1994; Francis and Hennessey, 1995; Kohidai, 1995; Kuruvilla et al, 1997; Hennessey, 2005; Leick and Lindemose, 2007; Rodgers et al, 2008) and Paramecium (Van Houten, 1978, 1990; Clark et al, 1992; Hennessey et al, 1995; Kim et al, 1997; deOndarza et al, 2003; Bell et al, 2007; Valentine et al, 2008), although the possible involvement of GPCRs and G‐proteins in these responses have not yet been established.…”

“…Since PP (proteose peptone) is known to be a chemoattractant in Tetrahymena (Kohidai, 1985; Hellung‐Larsen et al, 1986; Koppelhus et al, 1994; Leick et al, 1994), and some nutrient receptors in animal cells respond to both hydrolysed peptides (like those in proteose peptone) and lysophospholipids, e.g. like LPA (lysophosphatidic acid) (Choi et al, 2007), we became interested in LPA as a possible chemoattractant.…”

Motility and Energy Taxis of Salmonella spp.

“…A second method is a capillary assay. In this assay, heparinized glass capillaries bridge between a bacterial suspension and a test solution (Koppelhus et al, 1994;Leick & Helle, 1983). Two-phase assays in spectrophotometer plastic cuvettes are suitable to monitor the chemotaxis-mediated migration between the two fluid phases (Koppelhus et al, 1994).…”

“…In this assay, heparinized glass capillaries bridge between a bacterial suspension and a test solution (Koppelhus et al, 1994;Leick & Helle, 1983). Two-phase assays in spectrophotometer plastic cuvettes are suitable to monitor the chemotaxis-mediated migration between the two fluid phases (Koppelhus et al, 1994). The T-maze assay allows the quantification of the chemoresponse of two substances in direct comparison, using a T-shaped experimental arrangement of three containers (Van Houten et al, 1982).…”