Abstract
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.
Highlights
Basal bodies are cylindrical microtubular organelles that form the base of eukaryotic flagella and are structurally similar to centrioles (Tassin and Bornens, 1999)
Centrin deficiency reduced the number of flagella and bbs Chlamydomonas reinhardtii was transformed with a plasmid containing a sense-antisense hybrid construct of partial centrin DNA under the control of the strong constitutive HSP70A/ rbcS2-fusion promoter (Fig. 1A)
In wild-type cells, centrin is present both in the distal connecting fiber (dCF) interconnecting the two flagellar-bearing bbs and in the nucleus-bb connectors (NBBCs) that are surrounded by thin centrin fimbriae and link the bbs to the cell nucleus (Salisbury et al, 1988) (Fig. 1Ca, Fig. 5Aa′)
Summary
Basal bodies (bbs) are cylindrical microtubular organelles that form the base of eukaryotic flagella and are structurally similar to centrioles (Tassin and Bornens, 1999). Two pathways of bb/centriole assembly can be distinguished: the de novo pathway and the templated pathway (Marshall et al, 2001). The latter term refers to a process in which new bbs form once per cell cycle in the proximity of pre-existing ones; during cell division, pairs of bbs/centrioles are distributed semi-conservatively (Kochanski and Borisy, 1990). Controlled assembly of bbs and their proper segregation during mitosis are prerequisites to maintaining a constant number of this cell organelle. Centrioles/bbs are usually surrounded by a more-or-less structured matrix known as the pericentriolar material, and both together form the centrosome, which is the dominant microtubuleorganizing center (MTOC) in eukaryotic cells. Control of centriole number seems to be important for genome stability (Winey, 1996)
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