GFP as a tool for the analysis of proteins in the flagellar basal apparatus of Chlamydomonas.

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.

Centrin deficiency in Chlamydomonas causes defects in basal body replication, segregation and maturation.

Centrin, a 20 kDa calcium-binding protein, is a constituent of contractile basal body-associated fibers in protists and of various centrosomal structures. A construct inducing centrin RNAi was used to study the effect of centrin deficiency in Chlamydomonas. Transformants contained variable amounts of residual centrin (down to 5% of wild-type) and lacked centrin fibers. They displayed a variable flagellar number phenotype with mostly nonflagellate cells, suggesting that centrin is required for basal body assembly. Furthermore, basal bodies often failed to dock to the plasma membrane and to assemble flagella, and displayed defects in the flagellar root system indicating that centrin deficiency interferes with basal body development. Multiple basal bodies caused the formation of additional microtubular asters, whereas the microtubular cytoskeleton was disordered in most cells without basal bodies. The number of multinucleated cells was increased, indicating that aberrant numbers of basal bodies interfered with the cytokinesis of Chlamydomonas. In contrast to wild-type cells, basal bodies in centrin-RNAi cells were separated from the spindle poles, suggesting a role of centrin in tethering basal bodies to the spindle. To test whether an association with the spindle poles is required for correct basal body segregation, we disrupted centrin fibers in wild-type cells by over-expressing a nonfunctional centrin-GFP. In these cells, basal bodies were disconnected from the spindle but segregation errors were not observed. We propose that basal body segregation in Chlamydomonas depends on an extranuclear array of microtubules independent of the mitotic spindle.