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

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 wildtype) 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 wildtype 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.

Gametogenesis in the Chlamydomonas reinhardtii minus Mating Type Is Controlled by Two Genes, MID and MTD1

“…RNAi demonstrates an essential role for MTD1 in minus gametogenesis: MTD1 function was evaluated using RNAi (Sineshchekov et al 2002;Koblenz et al 2003). A hairpin RNAi plasmid containing inverted pairs of the third exon of MTD1, with the third intron serving as the middle loop, was driven by the constitutive HSP70A/rbcS2 promoter ( Figure 5A) (Schroda et al 2000;Koblenz et al 2003).…”

“…A hairpin RNAi plasmid containing inverted pairs of the third exon of MTD1, with the third intron serving as the middle loop, was driven by the constitutive HSP70A/rbcS2 promoter ( Figure 5A) (Schroda et al 2000;Koblenz et al 2003). This RNAi construct was cotransformed into wild-type mt À cells with pSI103.…”

Centrin Gene Disruption Impairs Stage-specific Basal Body Duplication and Cell Cycle Progression in Leishmania

“…Several mechanisms can explain failed cell division. Basal body/centriole duplication occurs once/cell division, and failed duplication or overproduction of these organelles would lead to aberrant mitotic spindles (2,4,29). The exact mechanism by which centrin affects basal body duplication in eukaryotic cells including the amastigote cells of Leishmania remains unknown.…”

“…Centrin has been characterized in eukaryotes from unicellular organisms such as Chlamydomonas (1,2) to humans (3)(4)(5). Fibers composed of centrin and its binding protein Sfi1p act as contractile or elastic connections between centrioles/basal bodies and other elements of the centrosome to mediate dynamic changes in its overall structure (6,7).…”

“…Similarly, silencing of human centrin 2 (HsCEN2) in HeLa cells impaired centriole duplication and resulted in the failure of cytokinesis (4). A centrin-deficient mutant of Chlamydomonas was nonflagellate because of defects in the flagellar root system (2). Abnormal centrosomes were noticed in human tumor cells because of the presence of excess levels of many centrosome proteins including centrin (11).…”