title: GFP as a tool for the analysis of proteins in the flagellar basal apparatus of Chlamydomonas.
abstract: Green fluorescent protein (GFP) was used to analyse three proteins in the flagellar basal apparatus of C. reinhardtii: (1) Striated fiber assemblin (SFA), the major component of the striated microtubule-associated fibers; (2) Centrin, present in the nucleus basal body connectors (NBBCs) and the distal connecting fiber (dCF) between the two basal bodies; and (3) DIP13, the Chlamydomonas homologue of human autoantigen NA14. The fusions co-localized with the wild-type proteins when expressed moderately. Overexpression of centrin-GFP and DIP13-GFP resulted in the formation of large aggregates and disturbed the distribution of the respective wild-type proteins. The amount of wild-type DIP13 was significantly reduced in cells overexpressing DIP13-GFP. Moreover, the cells frequently failed to assemble full-length flagella and flagellar regeneration was delayed, indicating a role of DIP13 during flagellar assembly. In contrast, overexpression of GFP-SFA, which retained more wild-type properties than SFA-GFP, increased the size of the striated fibers without altering the cross-shaped pattern. Abnormal patterns were observed in centrin-deficient cells, suggesting that centrin is required for proper localization of SFA. Photobleaching of GFP-SFA fibers indicated that GFP-SFA in the fibers is turned over slowly. Conditionally expressed centrin-GFP was incorporated into NBBCs in regions close to the basal bodies, but underrepresented in the dCF, indicative of a different dynamic of these two centrin fibers. Bending of the NBBCs was observed in vivo during flagellar motion, indicating that the filaments are flexible. In conclusion, in Chlamydomonas GFP-tagging is a useful tool for yielding new insights into the function and properties of the analyzed proteins.
authors.affiliation: Department of Botany, University of Cologne, Cologne, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Schoppmeier
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Mages
authors.forename: Karl-Ferdinand
authors.initials: KF
authors.lastname: Lechtreck
mesh_terms: D020418:Algal Proteins / Q000096:biosynthesis*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000096:biosynthesis*; D016825:Chlamydomonas reinhardtii / Q000378:metabolism / Q000648:ultrastructure*; D002868:Chromosomal Proteins, Non-Histone / Q000096:biosynthesis*; D005407:Flagella / Q000502:physiology* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012038:Regeneration / Q000502:physiology; D012995:Solubility
chemical_list: D020418:Algal Proteins; D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; C096668:striated fiber assemblin; C056608:caltractin; D049452:Green Fluorescent Proteins