IC2 participates in the cooperative activation of outer arm dynein densely attached to microtubules.

Ciliary outer-arm dynein (OAD) consists of heavy chains (HCs), intermediate chains (ICs), and light chains (LCs), of which HCs are the motor proteins that produce force. Studies using the green alga Chlamydomonas have revealed that ICs and LCs form a complex (IC/LC tower) at the base of the OAD tail and play a crucial role in anchoring OAD to specific sites on the microtubule. In this study, we isolated a novel slow-swimming Chlamydomonas mutant deficient in the IC2 protein. This mutation, E279K, is in the third of the seven WD repeat domains. No apparent abnormality was observed in electron microscope observations of axonemes or in SDS-PAGE analyses of dynein subunits. To explore the reason for the lowered motility in this mutant, in vitro microtubule sliding experiments were performed, which revealed that the motor activity of the mutant OAD was lowered. In particular, a large difference was observed between wild type (WT) and the mutant in the microtubule sliding velocity in microtubule bundles formed with the addition of OAD: ~35.3 µm/sec (WT) and ~4.3 µm/sec (mutant). From this and other results, we propose that IC2 in an OAD interacts with the ß HC of the adjacent OAD, and that an OAD-OAD interaction is important for efficient beating of cilia and flagella.Key words: cilia, axoneme, dynein heavy chain, cooperativity.

Release of Sticky Glycoproteins from Chlamydomonas Flagella During Microsphere Translocation on the Surface Membrane.

Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) attached to the flagellar membrane move bidirectionally along the flagellum. This surface motility enables cells to glide on a solid substrate to which they are attached by the flagellar surface. Previous studies suggested that microsphere movement and gliding motility result from the movement of transmembrane glycoprotein(s) within the plane of the plasma membrane, driven by intraflagellar transport (IFT), which utilizes cytoplasmic dynein and kinesin-2. However, it is not well understood how a cell can continuously glide in one direction further than a single flagellar length. Here we show that, during microsphere translocation on the flagella of a non-motile mutant, pf18, some flagellar glycoproteins, including FMG-1B and FAP113, detach from the membrane and attach to the microspheres. We propose that such relocation of surface glycoproteins underlies the ability to glide over a long distance. Surface motility is likely common to cilia/flagella of various organisms, as a similar microsphere movement is observed in the apical ciliary tuft in sea urchin embryos.

Extremely low polymerizability of a highly-divergent Chlamydomonas actin (NAP).

Novel actin-like protein (NAP) is a highly divergent actin expressed in Chlamydomonas. With its low sequence similarity, it is uncertain whether NAP can polymerize into filaments. Here I assessed it by ectopically expressing enhanced green fluorescent protein-tagged NAP (EGFP-NAP) in cultured cells. EGFP-NAP was excluded from stress fibres but partially co-localized with endogenous actin in the cell periphery. In fluorescence recovery after photobleaching experiment, turnover rate of EGFP-NAP was similar to the estimated diffusion rate of monomeric actin. Therefore, EGFP-NAP likely accumulates by diffusion. These findings suggest that NAP has extremely poor ability to polymerize.

Chlamydomonas reinhardtii tubulin-gene disruptants for efficient isolation of strains bearing tubulin mutations.

The single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two ß-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we screened an insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. Most of the disruptants did not exhibit major defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate tubulin-mutants resistant to the anti-tubulin agents propyzamide (pronamide) or oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 12 strains bearing 7 different ß-tubulin mutations. One of the mutations is at a residue similar to that of a mutation site known to confer drug resistance in human cancer cells. Some strains had the same amino acid substitutions as those reported previously in C. reinhardtii; however, the mutants with single tubulin genes showed slightly stronger drug-resistance than the previous mutants that express the mutated tubulin in addition to the wild-type tubulin. Such increased drug-resistance may have facilitated sensitive detection of tubulin mutation. Single-tubulin-gene disruptants are thus an efficient background of generating tubulin mutants for the study of the structure-function relationship of tubulin.

Impaired flagellar regeneration due to uncoordinated expression of two divergent actin genes in Chlamydomonas.

Chlamydomonas has two actin genes: one encoding a conventional actin (90% amino acid identity with mammalian actin) and the other a highly divergent actin (NAP; 64% identity). The expression of the two genes is regulated in a mutually exclusive manner. Thus, ida5, a mutant that lacks the conventional actin (CrA) gene, expresses NAP abundantly, whereas wild-type cells express NAP only negligibly under normal conditions. To explore the physiological significance of the two actins, chimeric genes with the 5' upstream region of one gene replaced by that of the other were constructed and used to transform ida5. The transformant (TF5) with a chimeric clone comprising the 5'-untranslated region from the NAP gene and the CrA-encoding sequence recovered the dyneins missing in ida5 and showed almost normal motility. After deflagellation of this transformant, however, only about 30% of cells grew flagella, unlike wild-type cells, >80% of which displayed reflagellation. Transformant TF10, which contains the CrA upstream region and NAP coding region, underwent reflagellation normally, as did the parent strain, ida5. In TF5, the mRNA level of both CrA and NAP was increased greatly during reflagellation. In light of the recent finding that NAP mRNA is expressed transiently upon reflagellation in wild-type cells, the described results suggest that 1) the expression of NAP mRNA is indispensable for flagellation and 2) robust expression of CrA may inhibit proper flagellation by interfering with the function of NAP in the early stages of reflagellation.

Identification of a novel actin-related protein in Tetrahymena cilia.

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In eukaryotes, its gene belongs to a highly conserved gene family, while the protein is also a member of an actin superfamily comprising various kinds of actin-related proteins (Arps). A ciliate, Tetrahymena, has a unique conventional actin. Data from the TIGR Tetrahymena genome project and our own research suggest the existence of 12 actin-like sequences: four conventional actins, two of Arp4, one each of Arp1, Arp2, Arp3, Arp5, and Arp6, and a novel actin-related protein, tArp. We cloned the entire cDNA sequences of Tetrahymena Arp2 (tArp2), Tetrahymena Arp3 (tArp3), and tArp for the work described herein. In phylogenetic analyses, tArp was not included in any Arp subfamily. Unlike other known Arps, tArp localizes in cilia, and its expression was upregulated after deciliation. To see the precise localization of tArp, cilia were fractionated and analyzed using specific antibodies. tArp was observed preferentially in the "outer-doublet" fraction, while actin was found in the "crude-dynein" fraction. In immunoelectron microscopy, most of the gold particles were found either on the outer-doublet or central-pair microtubules. These results suggest that tArp is a ciliary component and that it has a unique function in the formation and maintenance of cilia.

A novel family of unconventional actins in volvocalean algae.

The unicellular green alga Chlamydomonas reinhardtii has two actin genes, one encoding a conventional actin (90% amino acid identity with mammalian actin), the other a highly divergent actin (64% identity) named novel actin-like protein (NAP). To see whether the presence of conventional and unconventional actins in a single organism is unique to C. reinhardtii, we searched for genomic sequences related to the NAP sequence in several other species of volvocalean algae. Here we show that Chlamydomonas moewusii and Volvox carteri also have, in addition to a conventional actin, an unconventional actin similar to the C. reinhardtii NAP. Analyses of the deduced protein sequences indicated that the NAP homologues form a distinct group derived from conventional actin.

Expression of conventional and unconventional actins in Chlamydomonas reinhardtii upon deflagellation and sexual adhesion.

Chlamydomonas has two actin genes, one coding for a conventional actin and the other coding for a highly divergent actin. The divergent actin NAP (for "novel actin-like protein") is expressed only negligibly in wild-type cells but abundantly in a null mutant of conventional actin, the ida5 mutant. The presence of the dormant NAP gene suggests that NAP may also have its own function in wild-type cells under some conditions. However, no specific functions have been suggested. In this study, we examined the expression of actin and NAP in wild-type and ida5 cells under conditions where actin function has been shown to be important. We found that deflagellation induces the expression of NAP as well as that of actin in wild-type cells. The expressed NAP becomes localized to the regrown flagella, apparently without being associated with dynein. Mating of gametes also increased the expression of actin in wild-type cells and that of NAP in ida5 cells, resulting in accumulation of these proteins in flagella (in both wild-type and ida5 cells) and the fertilization tubule (only in wild-type cells). However, it did not induce significant NAP expression in wild-type cells. These and other observations suggest that the expression of actin and NAP mRNAs is controlled by two discrete mechanisms and that NAP plays a role in flagellar formation in wild-type cells.