Deflagellation and Regeneration in Chlamydomonas

Eukaryotic cilia/flagella are one of the only cellular structures that can be removed without injuring cells, can be highly purified for biochemical analysis, and, in many cells, can be completely reassembled within 90 minutes. Following amputation, the expression of many flagellar genes is up-regulated, and many are packaged and associated with intraflagellar transport (IFT) particles for transport to flagellar bases and into growing flagella. Studies of deciliation and ciliary growth provide insight to mechanisms that regulate microtubule assembly and length, mechanisms that regulate the transport of soluble cytoplasmic proteins into the ciliary compartment and their assembly into microtubules, and mechanisms that regulate trafficking of membrane proteins and lipids to the plasma membrane or to ciliary bases and their movement into and out of the cilium. These are important for motility and for signal transduction.

Deciliation methods for many cells have been developed and most require extracellular calcium ions and activation of signaling pathways that regulate microtubule severing (Quarmby, 2009). Deciliation occurs at the distal end of the basal bodies and, as soon as axonemes are severed, the membrane reseals and basal bodies begin to regenerate cilia.

Chlamydomonas is an ideal organism with which to study ciliary regeneration. Cells are easily and inexpensively cultured, flagellar amputation and regeneration is uniform in all cells in a population and growth can be assayed by observing fixed or living cells with a phase contrast microscope equipped and a 40x objective lens. Flagellar regeneration on individual living cells can be observed using paralyzed mutants immobilized in agarose. Because deflagellation leaves cells intact, the released flagella can be purified without contamination with cellular debris. The most reliable deciliation and regeneration method is the pH shock method developed by Reference 5 (also see References 4 and 11). Other methods are reviewed by Quarmby, (2009). The pH shock method is primarily used for Chlamydomonas but can be used for deciliation and regeneration of Tetrahymena cilia (Gaertig et al., 2013).