The Chlamydomonas VFL3 gene is essential for normal segregation and positioning of basal bodies
Carolyn D. Silflow, Brian P. Piasecki, Kelly R.B. Iyadurai, and Matthew Lavoie
Dept. of Plant Biology, St. Paul, MN 55108
Positioning and segregation of basal bodies (centrioles) in Chlamydomonas depend on a system of microtubule rootlets and other fibers in the basal body apparatus. Cells with a vfl3 mutation have a variable number of flagella and basal bodies with both spatial and rotational positioning defects. The VFL3 gene encodes a 65 kD basic coiled-coil protein, putative homologs of which are found in the genomes of the majority of flagellated eukaryotes including the moss Physcomitrella and humans. Results from immunofluorescence and immunogold labeling experiments localized Vfl3p to the lateral striated fibers. These fibers associate closely with probasal bodies and may assist in their rotational positioning. Basal bodies and probasal bodies in vfl3 mutant cells were visualized with an HA-tagged Uni2 protein which appears in M phase on newly forming probasal bodies. Many interphase vfl3 cells show evidence of templated basal body duplication, based on the appearance of diamond-shaped clusters of Uni2 protein staining. Up to 20% of vfl3 cells in interphase lack Uni2 staining. Some mutant cells are blocked or delayed in mitosis and some basal bodies fail to segregate normally to the spindle poles. The results suggest a role for Vfl3p in assembly and/or segregation of probasal bodies during cell division.
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