A CDK-related kinase regulates the length and assembly of flagella in Chlamydomonas

Abstract

Little is known about how cells regulate the size of their organelles. In this study, we find that proper flagellar length control in Chlamydomonas reinhardtii requires the activity of a new member of the cyclin-dependent kinase (CDK) family, which is encoded by the LF2 (long flagella 2) gene. This novel CDK contains all of the important residues that are essential for kinase activity but lacks the cyclin-binding motif PSTAIRE. Analysis of genetic lesions in a series of lf2 mutant alleles and site-directed mutagenesis of LF2p reveals that improper flagellar length and defective flagellar assembly correlate with the extent of disruption of conserved kinase structures or residues by mutations. LF2p appears to interact with both LF1p and LF3p in the cytoplasm, as indicated by immunofluorescence localization, sucrose density gradients, cell fractionation, and yeast two-hybrid experiments. We propose that LF2p is the catalytic subunit of a regulatory kinase complex that controls flagellar length and flagellar assembly.

Figure 1.

Flagellar phenotype of the lf2-6 mutant. (A–C) DIC images of WT cells with pairs of equal length flagella ∼13–14 μm long (A), an lf2-3 cell with long flagella ∼21 μm in length (B), and lf2-6 cells with unequal length flagella and stumpy flagella that have distal swollen tips (C; arrows). (D) lf2-6 cells were unable to regrow their flagella beyond a short stump even 5 h after flagellar amputation. (E and F) In EM thin sections, short aggregates of IFT-like particles (arrows) were found filling the space between the flagellar membrane and microtubules in these distal swellings. Bars (D), 10 μm; (E and F) 100 nm. (G) A histogram showing the total length of each pair of flagella in 50 lf2-6 cells. The length of the longer flagella was stacked on top of that of the shorter flagella.

Figure 2.

Structure, ClustalW multiple alignment, and RNA analysis of the LF2 gene. (A) Restriction map of the genomic region around the site of insertion in lf2-6. The restriction sites XhoI (X), Smal (S), and NotI (N) are shown. (B) C. reinhardtii LF2p is compared with a rat CDK2 and a rat PNQALRE (GenBank/EMBL/DDBJ accession no. Q63699 and Q4KM34). Identical and similar amino acids are shaded in black and gray, respectively. The 11 subdomains characteristic of serine/threonine kinases are delineated. Glycine (black dots) in the GXGXXG motif for ATP binding, the invariant lysine (asterisk) conserved in all functional kinases, residues corresponding to the cyclin-binding PSTAIRE motif (underlined), and the threonine (double underlined) that has to be phosphorylated for full activation of many CDKs are noted. (C and D) 25 μg of total RNA isolated from WT (CC-620) and various lf mutant strains at different times after pH-induced deflagellation in WT cells was size fractionated on formaldehyde agarose gels and transferred to nylon membranes. The blots were hybridized successively to a PCR probe spanning nucleotides 602–900 of the LF2 cDNA sequence, a genomic DNA probe of PF20 encoding a protein in the axonemal central pair, and a DNA probe from CRY1 (encodes ribosomal S14 protein) for loading controls.

Figure 3.

Western blot analysis of HA-tagged LF2 protein. (A) Total protein from two strains (2C11 and 2A10) rescued with the untagged LF2 construct and four strains (3C1, 3F1, 3G1, and 3A2) rescued with the HA-tagged LF2 construct were size fractionated on an SDS-PAGE gel, transferred to a PVDF filter, and detected with an HA antibody (top). A cluster of bands migrating at ∼42 kD were observed only with cells expressing the tagged construct. When total cell protein from 3G1 was resolved on a longer gel (bottom; 15- and 60-s exposures), at least three isoforms were visible (arrows). (B) Soluble proteins were extracted from 3F1 cells by freezing/thawing twice in a buffer either with (+PPI) or without phosphatase inhibitors (no PPI). Cell extracts were incubated at 37°C for the amount of time indicated and analyzed on a Western blot. (C) 2D gel analysis of LF2p. Arrows point to the three major isoforms of HA-LF2p. (D) Comparison of the amount of LF2p in whole cells, cell bodies, flagella, and axonemes. Flagellar or axonemal protein from 10⁶ (1×), 5 × 10⁶ (5×), and 3.6 × 10⁷ (36×) cells were compared with 10⁶ whole cells and cell body proteins from 10⁶ (1×), 10⁵ (0.1×), and 10⁴ (0.01×) cells. The same blot was reacted with an antibody to a chloroplast protein, OEE1, to determine the amount of cell body contamination in the flagella preparations.

Figure 4.

LF2p localized with LF3p. (A) Immunofluorescence studies localized HA-LF2p and HA-LF3p to punctate spots inside the cells. Top panels are DIC images, and bottom panels are immunostaining with the HA antibody and an AlexaFluor488 secondary antibody. (B) LF2p comigrated closely with LF3p in sucrose density gradients. Cell extracts from strains expressing either HA-LF2p or HA-LF3p were mixed and loaded on a 5–20% sucrose density gradient for parallel analysis. Samples were collected from top to bottom and analyzed using SDS-PAGE and Western blots. In the bottom panel, the sedimentation of LF4p was compared with that of LF2p.

Figure 5.

Genetic lesions in four lf2 mutants. (A) Nucleotide changes in the different alleles are indicated under the WT genomic sequences. Three of the mutations involve intron splice sites. The normal splice sites (arrowheads) and alternative splice sites (double arrows) are indicated. The corresponding changes in protein sequences are shown in gray. (B) Summary of the protein structure and phenotype of the mutants.

Figure 6.

Phenotype of K41R- and G21V-rescued cells. (A) Histograms showing flagellar length distribution in populations of lf2-3 and lf2-6 mutant cells and lf2-6 cells rescued with the WT construct or mutant constructs (K41R and G21V). (B) Kinetics of flagellar regeneration in WT cells, lf2-3 mutant, and strains transformed with the mutant LF2 constructs. Cells harboring the K41R construct showed severe retardation in flagellar regeneration similar to lf2-3 cells. (C) Western blot analysis of transformants rescued with the WT or K41R construct. Three isoforms of LF2p were detected in WT transformants, but only the fastest migrating form of LF2p was observed in K41R transformants as well as in G21V transformants (not depicted).