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

Abstract

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.

Turnover of Actin in Chlamydomonas Flagella Detected by Fluorescence Recovery After Photobleaching (FRAP)

“…Those motile cells displayed rhodamine fluorescence in their flagella (data not shown). These observations are essentially the same as reported by Hayashi et al (2001). …”

“…Upon incorporation of exogenous actin, it became motile due to the recovery of missing inner arm dyneins. As in our previous study (Hayashi et al, 2001), electroporation at ~1200 V/cm was found to be optimal, where about 50% of total cells were killed and 10-20% of surviving cells regained motility. Those motile cells displayed rhodamine fluorescence in their flagella (data not shown).…”

“…A problem in applying FRAP techniques to Chlamydomonas has been that it is difficult to introduce fluorescent proteins, or to express GFP-conjugated proteins. Recently, however, our laboratory has developed a method to introduce exogenous proteins into live Chlamydomonas cells using electroporation, and has shown that fluorescently labeled actin is functionally incorporated into inner arm dyneins in a mutant lacking the conventional actin gene (Hayashi et al, 2001). Using this technique, we carried out the first attempt to examine the dynamic exchange of actin in Chlamydomonas flagella.…”

“…Fluorescent monomeric actin was introduced into ida5oda1 cells using electroporation, following the method of Hayashi et al (2001). Briefly, cells were treated with autolysin to remove the cell walls, and subjected to electroporation in the presence of 0.1 mg/ ml labeled actin, 0.2 mM ATP-imidazole (pH 7.4), 0.1 mM CaCl2, 0.5 mM 2-mercaptoethanol, and 60 mM sucrose.…”

“…Actin-incorporated cells displayed flagellar motility and fluorescence. The amount of actin introduced was apparently sufficient for the cell to recover all actin-containing dyneins, since the ida5oda1 cells that became motile after electroporation displayed swimming velocities indistinguishable from those of oda1 cells (Hayashi et al, 2001). Electroporated cells were kept overnight under dark conditions until used in photobleach experiments.…”