Nucleus‐basal body connector in Chlamydomonas: Evidence for a role in basal body segregation and against essential roles in mitosis or in determining cell polarity

Abstract

In the unicellular biflagellate green alga Chlamydomonas reinhardtii each basal body is linked to the nucleus by a fibrous nucleus-basal body connector (NBBC) that contains the calcium-binding protein centrin. (Wright et al.: Journal of Cell Biology 101:1903-1912.; Salisbury et al.: Journal of Cell Biology 107:635-642; Huang et al.: Journal of Cell Biology 107:121-131). In order to explore the cellular function of the NBBC we used antiserum directed against centrin to examine a number of mutants known to be defective for basal body assembly and/or localization. Of three variable flagella-number mutants examined, one, vfl-2, is dramatically defective with respect to the NBBC in that 1) the union between basal bodies and nucleus is very labile, 2) there is no detectible centrin in the NBBC region, and 3) total cellular centrin levels are reduced 75-80% relative to wild type. The existence of these defects in a mutant incapable of maintaining normal flagellar number supports the view that the NBBC plays an important role in determining proper basal body localization and/or segregation. In contrast to vfl-2, the mutants vfl-1, vfl-3, uni-1, and bald-2 contain approximately normal levels of centrin and possess stable NBBCs. The observation of NBBCs in the mutant bald-2, which lacks all but very rudimentary basal bodies, indicates that the assembly of the NBBC does not require fully formed basal bodies and that such assembly may not require basal bodies at all. Finally, the possibility that the NBBC is required for induction of gene expression following deflagellation was tested by examining vfl-2 for such induction. Results indicate that the connector does not play a necessary role in the induction process.

Centrin deficiency inChlamydomonascauses defects in basal body replication, segregation and maturation

“…In wild-type cells, centrin and bbs are located at the poles of the spindle which, in Chlamydomonas, is intranuclear with fenestrae at the poles (e.g. Johnson and Porter, 1968;Coss, 1974;Wright et al, 1989) (Fig. 4Ba-c).…”

“…4Ba-c). It was assumed that, in Chlamydomonas vfl2, proper segregation of bbs fails due to the absence of intact NBBCs (Wright et al, 1989). Thus, we determined the position of bbs with respect to the mitotic spindle in centrin-depleted cells.…”

“…Our data indicate significant differences in the phenotype of vfl2 cells comprising a centrin point mutation and the effect of centrin knockdown. Vfl2 cells contain 20-25% of residual centrin (Wright et al, 1989), whereas strains with down to 5% of residual centrin were identified using centrin-RNAi. In all strains analyzed in detail, the centrin-RNAi effect was lost after prolonged cultivation and wild-type centrin structures re-appeared, excluding the possibility that the observed phenotypes were caused by insertional mutagenesis of other genes.…”

“…Moreover, silencing of human centrin 2 impairs centriole duplication in HeLa cells, emphasizing that centrin is required for the replication of bbs and centrioles (Salisbury et al, 2002). A point mutation in the single-copy centrin gene of the biflagellate green alga Chlamydomonas causes the vfl2 phenotype (for 'variable flagellar number'), suggesting that centrin is also involved in the segregation of bbs (Wright et al, 1989). In Chlamydomonas, centrin is located in the stellate structure of the flagellar transition region, which represents the border between the bb and the axoneme, in the distal connecting fiber (dCF) between bbs, and in the two nucleusbb connectors (NBBCs), which link the bbs to the nucleus (Salisbury, 1995).…”

“…In Chlamydomonas, centrin is located in the stellate structure of the flagellar transition region, which represents the border between the bb and the axoneme, in the distal connecting fiber (dCF) between bbs, and in the two nucleusbb connectors (NBBCs), which link the bbs to the nucleus (Salisbury, 1995). These structures are defective in vfl2 cells and it has been assumed that the missing link between bbs and the nucleus causes mistakes in bb segregation during cell division (Wright et al, 1989). Thus, centrin seems to be involved in both the production of bbs and their distribution to daughter cells, raising the question of how centrin performs these multiple tasks.…”