Turnover of actin in Chlamydomonas flagella detected by fluorescence recovery after photobleaching (FRAP).
Recent indirect observations have suggested that various axonemal proteins in cilia and flagella of live cells undergo turnover independently of shortening or elongation of the axoneme. To gain direct evidence, here we examined using a FRAP (fluorescence recovery after photobleaching) technique whether actin, a subunit of inner arm dynein, is being turned over in Chlamydomonas flagella. Fluorescently labeled rabbit actin was introduced by electroporation into the cells of ida5oda1, a double mutant between oda1 lacking outer arm dynein and ida5 lacking several species of inner arm dyneins due to the absence of a conventional-type actin. In actin-loaded cells, flagella became motile and fluorescent due to incorporation of inner-arm dyneins containing the labeled actin. Cells were sandwiched between an agar layer and a coverslip so as to restrict flagellar movement. After a small portion of a flagellum was photobleached, the fluorescence intensity in the bleached area was monitored with a sensitive video camera. The fluorescence intensity in the photobleached region was found to recover 10-40% of the original level over several tens of minutes without changing its position. The time course and extent of the recovery varied greatly from one cell to another, suggesting that the turnover depends on cellular conditions. Western blot analysis indicated that 70-80% of flagellar actin was associated with the axoneme. Hence this experiment provides direct evidence that an axonemal component undergoes dynamic exchange in stationary flagella.
Watanabe
Yumi
Y
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
Hayashi
Masahito
M
Yagi
Toshiki
T
Kamiya
Ritsu
R
D000199:Actins / Q000378:metabolism*; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D005407:Flagella / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D009154:Mutation / Q000235:genetics; D011817:Rabbits
D000199:Actins