Chapter 1: Flagellar Amputation and Regeneration in Chlamydomonas

In flagellar autotomy, Chlamydomonas cells shed their flagella in response to stressful conditions, including extremes of temperature or pH and the presence of noxious agents such as detergents or alcohols in the medium. It serves a protective function for cells in the environment—under adverse conditions the flagella are shed to minimize exposure to environmental stress. There is existence of two different signaling mechanisms for flagellar autotomy. One of these involves the influx of Ca²⁺ from the medium through specific channels; the other is proposed to be dependent on the release of intracellular Ca²⁺ stores. A rapid increase in inositol 1, 4, 5 triphosphate (IP₃) is associated with flagellar autotomy, probably acting to release intracellular calcium. In Chlamydomonas the regeneration of flagella has been studied to provide insight into the synthesis of proteins that constitute the flagella and to characterize mutants with defective flagellar assembly. The procedures described in this chapter are used to amputate flagella under conditions that allow continued cell viability and flagellar regeneration. The most widely used and adaptable procedure to any research or teaching laboratory is pH shock. Suction deflagellation presumably acts by pulling the flagella through the holes of a nitrocellulose filter while retaining the cells on the filter. Techniques have been developed to deflagellate small numbers of cells in each well of a 96-well culture dish. This helps identify mutants with defective flagellar regeneration. Wild-type cells begin to regenerate flagella after amputation. Flagellar length can be assayed by aligning the flagella on fixed cells with an ocular micrometer, which consists of a small glass disk that fits into the eyepiece of the microscope. The grid is calibrated by observing a field micrometer grid.