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 toward experimental manipulation of actin in dynein, we developed a method for introducing exogenous actin into Chlamydomonas cells by electroporation. A non-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 under optimal conditions. In addition, by taking advantage of their phototactic behavior, the rescued cells could be concentrated. Motility was also recovered with fluorescently labeled actin; in this case, axonemes became fluorescent 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 which no efficient methods have been developed for expressing or introducing foreign proteins and other macromolecules.
Hayashi
M
M
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan.
Hirono
M
M
Kamiya
R
R
D000199:Actins / Q000008:administration & dosage* / Q000172:deficiency / Q000493:pharmacokinetics; D000818:Animals; D002465:Cell Movement / Q000502:physiology; D016764:Cell Polarity; D002696:Chlamydomonas / Q000378:metabolism; D004398:Dyneins / Q000172:deficiency / Q000378:metabolism*; D004574:Electromagnetic Fields; D018274:Electroporation / Q000379:methods; D005407:Flagella / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis; D009132:Muscles / Q000737:chemistry*; D011817:Rabbits; D020127:Recovery of Function / Q000502:physiology; D012710:Serum Albumin, Bovine / Q000008:administration & dosage
D000199:Actins; D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine; D004398:Dyneins