Recovery of flagellar dynein function in a Chlamydomonas actin/dynein-deficient mutant upon introduction of muscle actin by electroporation

Flagellar and ciliary inner-arm dyneins contain actin as a subunit; however , the function of this actin subunit remains unknown. As a first step towar d experimental manipulation of actin in dynein, we developed a method for i ntroducing exogenous actin into Chlamydomonas cells by electroporation. A n on-motile mutant, ida5oda1, lacking inner-arm dyneins due to the absence of conventional actin, was electroporated in the presence of rabbit skeletal muscle actin. About 20% of the electroporated cells recovered motility unde r optimal conditions. In addition, by taking advantage of their phototactic behavior, the rescued cells could be concentrated. Motility was also recov ered with fluorescently labeled actin; in this case, axonemes became fluore scent after electroporation, suggesting that actin was in fact incorporated as a dynein subunit. The feasibility of incorporating a substantial amount of macromolecules by electroporation will be useful not only for studying actin function, but also for a variety of studies using Chlamydomonas in wh ich no efficient methods have been developed for expressing or introducing foreign proteins and other macromolecules. (C) 2001 Wiley-Liss, Inc.