Centrin deficiency in Paramecium affects the geometry of basal-body duplication.
BACKGROUND
Ciliary or flagellar basal bodies and centrioles share the same architecture and remarkable property of duplicating once per cell cycle. Duplication is known to proceed by budding of the daugther organelle close to and at right angles to the mother structure, but the molecular basis of this geometry remains unknown. Among the handful of proteins implicated in basal-body/centriole duplication, centrins seem required in all eukaryotes tested, but their mode of action is not clear. We have investigated centrin function in Paramecium, whose cortical organization allows detection of any spatial or temporal alteration in the pattern of basal-body duplication.


RESULTS
We have characterized two pairs of genes, PtCEN2a and PtCEN2b as well as PtCEN3a and PtCEN3b, orthologs of HsCEN2 and HsCEN3, respectively. GFP tags revealed different localization for the two pairs of gene products, at basal bodies or on basal-body-associated filamentous arrays, respectively. Centrin depletion induced by RNAi caused mislocalization of the neoformed basal bodies: abnormal site of budding (PtCen2ap) or absence of separation between mother and daughter organelles (PtCen3ap). Over successive divisions, new basal bodies continued to be assembled, but internalization of the mispositionned basal bodies led to a progressive decrease in the number of cortical basal bodies.


CONCLUSIONS
Our observations show that centrins (1) are required to define the site and polarities of duplication and to sever the mother-daughter links and (2) play no triggering or instrumental role in assembly. Our data underscore the biological importance of the geometry of the duplication process.
Ruiz
Françoise
F
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France. ruiz@cgm.cnrs-gif.fr
Garreau de Loubresse
Nicole
N
Klotz
Catherine
C
Beisson
Janine
J
Koll
France
F
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000172:deficiency* / Q000235:genetics; D002455:Cell Division / Q000502:physiology; D002502:Centrioles / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000172:deficiency* / Q000235:genetics; D003001:Cloning, Molecular; D016000:Cluster Analysis; D005407:Flagella / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010247:Paramecium / Q000378:metabolism* / Q000648:ultrastructure; D010802:Phylogeny; D034622:RNA Interference; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; C056608:caltractin; D049452:Green Fluorescent Proteins