The Vfl1 Protein in Chlamydomonas Localizes in a Rotationally Asymmetric Pattern at the Distal Ends of the Basal Bodies

Abstract

In the unicellular alga Chlamydomonas, two anterior flagella are positioned with 180° rotational symmetry, such that the flagella beat with the effective strokes in opposite directions (Hoops, H.J., and G.B. Witman. 1983. J. Cell Biol. 97:902–908). The vfl1 mutation results in variable numbers and positioning of flagella and basal bodies (Adams, G.M.W., R.L. Wright, and J.W. Jarvik. 1985. J. Cell Biol. 100:955–964). Using a tagged allele, we cloned the VFL1 gene that encodes a protein of 128 kD with five leucine-rich repeat sequences near the NH2 terminus and a large α-helical–coiled coil domain at the COOH terminus. An epitope-tagged gene construct rescued the mutant phenotype and expressed a tagged protein (Vfl1p) that copurified with basal body flagellar apparatuses. Immunofluorescence experiments showed that Vfl1p localized with basal bodies and probasal bodies. Immunogold labeling localized Vfl1p inside the lumen of the basal body at the distal end. Distribution of gold particles was rotationally asymmetric, with most particles located near the doublet microtubules that face the opposite basal body. The mutant phenotype, together with the localization results, suggest that Vfl1p plays a role in establishing the correct rotational orientation of basal bodies. Vfl1p is the first reported molecular marker of the rotational asymmetry inherent to basal bodies.

The Uni2 Phosphoprotein is a Cell Cycle–regulated Component of the Basal Body Maturation Pathway inChlamydomonas reinhardtii

“…The DNA was sequenced on both strands and the DNA sequences were assembled using Genetics Computer Group software (Devereux et al, 1984). Because no expressed sequence tag (EST) support for the gene was available, cDNA sequences were identified by using reverse transcriptase PCR amplification of UNI2 RNA as described by Silflow et al (2001). The 5Ј end of the RNA transcript extends at least 272 base pairs upstream of the putative start codon.…”

“…Pre-embedding immunogold EM of NFAps was performed as described by Silflow et al (2001). Ultrathin sections were imaged at (60,000ϫ) using a transmission electron microscope (TEM; CM-12; Philips) at 60 kV.…”

“…The UNI2 gene sequence is available at GenBank under the accession number EF675992. The pML9.7 plasmid was modified by inserting a sequence encoding three copies of the HA epitope (Silflow et al, 2001) into a BamHI site located 13 codons upstream of the stop codon to create the pML9.7-3xHA plasmid. Isolation of nucleic acids from Chlamydomonas and RNA blotting were carried out as described by Silflow et al (2001).…”

“…The bld2 mutation in the gene encoding -tubulin (Dutcher et al, 2002) and the uni3 mutation in the gene encoding ␦-tubulin (Dutcher and Trabuco, 1998) reveal the roles of these tubulins in assembly of B tubules and C tubules in triplet micro-tubules of the basal body wall. Mutations in the VFL1 gene encoding a basal body protein (Silflow et al, 2001) and in the VFL2 gene encoding centrin (Taillon et al, 1992) show the roles of these gene products in basal body positioning and segregation. Genomic and proteomic approaches have provided information on the protein composition of the flagella and basal bodies (Li et al, 2004;Keller et al, 2005;Pazour et al, 2005).…”

“…Ultrathin sections were cut and stained with 1% uranyl acetate in 50% methanol for 20 min at room temperature, followed by staining with lead citrate for 5 min. Sections were examined using a transmission electron microscope (CM-12; Philips, Mahwah, NJ) at 60 kV.Pre-embedding immunogold EM of NFAps was performed as described by Silflow et al (2001). Ultrathin sections were imaged at (60,000ϫ) using a transmission electron microscope (TEM; CM-12; Philips) at 60 kV.…”