Genetic and phenotypic analysis of flagellar assembly mutants in Chlamydomonas reinhardtii

Abstract

Conditional mutants for flagellar assembly (fla) provide a useful tool to study intraflagellar transport (IFT) at the molecular level, and provide a unique set of tools to analyze cilia. The analysis of IFT phenotypes of fla mutants at the permissive temperature by a quantitative image analysis approach identified four distinct phases of the IFT cycle and directly demonstrated structural and functional remodeling of IFT particles at both axonemal extremities. In addition, the genetic analysis of fla mutants reveal interesting interactions among genes involved in flagellar assembly that help to provide information about the structure and function of IFT particles and their motors. This chapter provides protocols to isolate, characterize, and identify conditional Chlamydomonas flagellar assembly mutants and their genes and to test genetic interactions among proteins encoded by these genes.

Fig. 1

(A) Kymographs of flagella from pf15, pf15; fla27, pf15; fla18, pf15; fla24, and pf15; fla9 cells. Vertical (y) and horizontal (x) axes represent distance from cell body (μm) and time of observation (s), respectively. Black and white arrows indicate traces formed by anterograde or retrograde IFT particles, respectively. The IFT phase that is defective in each recombinant is indicated (see Table III for further details). The strain pf15; fla9 and other double mutants (pf15; fla4, pf15; fla5, and pf15; fla21) behave similarly to each, and are not significantly different from pf15 at 21°C and are labeled as unknown (?). Velocity, frequency ratio, and overall particle frequency can be obtained from these images. (B, C) Kymographs of pf15 flagella analyzed by different methods. Kymograph in B was generated using the approach described previously (Iomini et al., 2001: Piperno et al., 1998). The kymograph in C was generated with MetaMorph (see text). Kymograph B has had noise removed while kymograph C has not. Lower levels of noise facilitate detection of particles trajectories. A bifurcation of a trajectory indicating a possible split of one particle or a sudden change in velocity of one of two particles moving together is resolved in B but not in C (*). Recordings of pf15 in B and C, and pf15; fla27, pf15; fla18, and pf15; fla24 in A are available as Supplemental movies 1, 2, 3, and 4, respectively (http://www.elsevierdirect.com/companions/9780123749734). Panel A is reprinted from Iomini et al. (2001).

Fig. 2

PCR products from meiotic progeny (1–9) of a cross of the mutant parent (P) by the CC-1952 (S1D1) strain. The primers span a repetitive GT region and the two strains have a size polymorphism. This marker is linked (9/9) to the mutant phenotype.

Fig. 3

Schematic diagram of SHIRT. SHIRT uses mapping and PCR-based markers to determine which parts of a BAC are responsible (cosegregate) for rescue of a mutant phenotype following transformation. (A) Diagram of three genes on a BAC from the mutant parent (black), the polymorphic mapping strain CC-1952 (red), and the BAC (blue). Primers for PCR are generally made to the 3′UTR of genes to be tested (arrows). (B) Bands of digested PCR products (dCAP markers) for the three genes from the three sources of DNA. The digested PCR products from the black and blue alleles cannot be distinguished from each other. (C) Three possible outcomes among NPD tetrads in which the mutant phenotype is observed in two of the four progeny (fla and +). The pattern with Gene 1 suggests that the BAC DNA is not responsible for rescue. The hybrid (Black and Blue) band indicates that the band is amplified from both the mutant and the BAC allele. The pattern with Gene 2 is consistent with the BAC DNA providing rescue. Further proof requires additional NPD tetrads with this pattern. The pattern with Gene 3 suggests that it is not integrated into the genome and therefore not responsible for rescue. This figure is reprinted from Iomini et al. (2009). (See Plate no. 6 in the Color Plate Section.)

Fig. 4

SHIRT analysis of the FLA15 transgene. PCR products from an NPD are shown. The parents (fla15 + BAC; CC-1952) are in lanes 1 and 2. The four meiotic progeny are in lanes 3–6. Lanes 3 and 6 are from cells aflagellate at 32°C and the band is consistent with the mutant allele. Lanes 4 and 5 are from flagellated cells and the bands are consistent with the wild-type FLA15 allele from the CC-1952 parent and the BAC contributed transgene.