Chlamydomonas mutants display reversible deficiencies in flagellar beating and axonemal assembly

Abstract

Axonemal complexes in flagella are largely pre-packaged in the cell body. As such, one mutation often results in the absence of the co-assembled components and permanent motility deficiencies. For example, a Chlamydomonas mutant defective in RSP4 in the radial spoke (RS), which is critical for bend propagation, has paralyzed flagella that also lack the paralogue RSP6 and three additional RS proteins. Intriguingly, recent studies showed that several mutant strains contain a mixed population of swimmers and paralyzed cells despite their identical genetic background. Here we report a cause underlying these variations. Two new mutants lacking RSP6 swim processively and other components appear normally assembled in early log phase indicating that, unlike RSP4, this paralogue is dispensable. However, swimmers cannot maintain the typical helical trajectory and reactivated cell models tend to spin. Interestingly the motile fraction and the spokehead content dwindle during stationary phase. These results suggest that 1) intact RS is critical for maintaining the rhythm of oscillatory beating and thus the helical trajectory; 2) assembly of the axonemal complex with subtle defects is less efficient and the inefficiency is accentuated in compromised conditions, leading to reversible dyskinesia. Consistently, several organisms only possess one RSP4/6 gene. Gene duplication in Chlamydomonas enhances RS assembly to maintain optimal motility in various environments.

A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex

“…Consistent with this, fewer cells expressing 1-316 swam in stationary phase cultures (axonemes were harvested from stationary phase cultures) than in the suspension from fresh plates or the log phase culture. This media effect on the motility level and assembly was noted previously in two spoke mutants Wei et al, 2010). The head protein deficiency in this strain missing RSP3's C terminus resembles the phenotype of the RSP2 mutant pf24 (Huang et al, 1981;Patel-King et al, 2004;, suggesting that both RSP2's and RSP3's C termini are involved in, albeit not required for, the assembly of the spoke head, and thus head proteins are not entirely absent when only one of them is defective.…”

“…The flagella of immotile cells were paralyzed or twitching. The mixture of swimmers and immotile cells from a single clone resembles the phenotype of several mutants with mild RS deficiencies (Huang et al, 1981;Gaillard et al, 2006;Wei et al, 2010). This indicates that 2-RSP3 polypeptides were restored to the axoneme of the swimmers and were capable of rescuing paralyzed flagella, albeit partially.…”

The DPY-30 Domain and Its Flanking Sequence Mediate the Assembly and Modulation of Flagellar Radial Spoke Complexes

“…Calmodulin was not probed because a change in the calmodulin content in axonemes with defective RSs is not expected to be evident against a background of this molecule from other axonemal complexes (16,18,43,61). The mixed population of motile and nonmotile cells in ⌬CT-Tag cultures is also a phenotype of pf26 allelic mutants that are defective in the gene encoding one of the two spokehead paralogues, RSP6 (13,63). The motile fraction in pf26 cultures decreases when the medium becomes exhausted, and the reduction was correlated with decreased spokehead assembly in axonemes (63).…”

“…The thinner stalk adheres to the 9 microtubule outer doublets of the axoneme, while the bulbous head intermittently contacts the central-pair (CP) apparatus (62). Defects in the RS result in a spectrum of motility deficiencies ranging from jerky flagella to reversible or full paralysis (63,66,67,69). It has been proposed that the periodic engagement between the RS and the CP enables sequential activation of dynein motors on the outer doublets that power rhythmic beating (62,66).…”

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii

“…4. Wild-type cells demonstrated the standard, wavy and helical-type paths with loop formation [34]. For lf2-5 and lf3-2, swim paths and waveforms were closest to those of WT, with swimming velocities being 70% and 88%, respectively, of the WT.…”