ASSAY OF CHLAMYDOMONAS PHOTOTAXIS

Phototaxis depends on slight light-dependent changes in the waveform of the cis and trans flagella, as revealed by step-up and step-down illumination of immobilized cells. Chlamydomonas responds phototactically to blue illumination with a peak response at 503 nm. The response can be either positive (cells swim toward light source) or negative (cells swim away from light source) depending on actinic beam intensity, history of cells' exposure to light, genetic background of cells, and other factors. The assay of phototaxis offers the most stringent test of Chlamydomonas flagellar function currently available, as it measures not only motility but also the ability of the flagella to respond to intracellular signals. This chapter describes a method for analysis of the swimming direction of a population of individual cells in response to light. The method has been particularly useful for the characterization of mutants with defects in their ability to phototax. The chapter also discusses culture and handling of Chlamydomonas cells and phototaxis room setup. Dark-adapted Chlamydomonas are quite light-sensitive. Therefore, the room must be lighttight when the door is closed to prevent biasing of light-dependent swimming. During the phototaxis assay, it is critical that the microscope objective be aligned with an imaginary line running between the outlets of the two fiberoptic cables so that the actinic light enters the field of view strictly from the left or right. There is description of microscopy and video equipment. Dark-field microscopy is preferred because the cells are then brightly illuminated against a dark background. Virtually any moderately sensitive video camera is acceptable except for cameras with noisy signals that lead to thresholding difficulties in the video processor; any monochrome monitor is also acceptable. The general procedure for data acquisition is examined. © 1995, Academic Press Inc.