Flagellar protein dynamics in Chlamydomonas.
Cilia and flagella appear to be stable, terminal, microtubule-containing organelles, but they also elongate and shorten in response to a variety of signals. To understand mechanisms that regulate flagellar dynamics, Chlamydomonas cells with nongrowing flagella were labeled with (35)S, and flagella and basal body components were examined for labeled polypeptides. Maximal incorporation of label into the flagella occurred within 3 h. Twenty percent of the flagellar polypeptides were exchanged. These included tubulins, dyneins, and 80 other axonemal and membrane plus matrix polypeptides. The most stable flagellar structure is the PF-ribbon, which comprises part of the wall of each doublet microtubule and is composed of tubulin and three other polypeptides. Most (35)S was incorporated into the high molecular weight ribbon polypeptide, rib240, and little, if any, (35)S is incorporated into PF-ribbon-associated tubulin. Both wild-type (9 + 2) and 9 + 0 flagella, which lack central microtubules, exhibited nearly identical exchange patterns, so labeling is not due to turnover of relatively labile central microtubules. To determine if flagellar length is balanced by protein exchange, (35)S incorporation into disassembling flagella was examined, as was exchange in flagella in which microtubule assembly was blocked by colchicine. Incorporation of (35)S-labeled polypeptides was found to occur into flagellar axonemes during wavelength-dependent shortening in pf18 and in fla10 cells induced to shorten flagella by incubation at 33 degrees C. Colchicine blocked tubulin addition but did not affect the exchange of the other exchangeable polypeptides; nor did it induce any change in flagellar length. Basal bodies also incorporated newly synthesized proteins. These data reveal that Chlamydomonas flagella are dynamic structures that incorporate new protein both during steady state and as flagella shorten and that protein exchange does not, alone, explain length regulation.
Song
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L
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Dentler
W L
WL
D000818:Animals; D002478:Cells, Cultured; D002696:Chlamydomonas / Q000737:chemistry*; D003078:Colchicine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000737:chemistry*; D015151:Immunoblotting; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D010455:Peptides; D003078:Colchicine